In situ-generated microfluidic assay structures, related kits, and methods of use thereof

ABSTRACT

In situ-generated microfluidic capture structures incorporating a solidified polymer network, methods of preparation and use, compositions and kits therefor are described. Microfluidic capture structures may be advantageously used for assays performed within the microfluidic environment, providing flexibility in assaying micro-objects such as biological cells. Assay reagents and analytes may be incorporated within the microfluidic capture structures.

This application is a non-provisional application claiming the benefitunder 35 U.S.C. 119(e) of U.S. Provisional Application No. 62/264,665,filed on Dec. 8, 2015, U.S. Provisional Application No. 62/333,821,filed on May 9, 2016, and of U.S. Provisional Application No.62/418,625, filed on Nov. 7, 2016, each of which disclosures is hereinincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

In biosciences and related fields, it can be useful to have the abilityto assay micro-objects within a microfluidic device. Some embodiments ofthe present disclosure include apparatuses and processes for in-situgeneration of microfluidic capture structures.

SUMMARY OF THE INVENTION

In one aspect, a microfluidic device for assaying micro-objects isprovided, including an enclosure having a substrate and microfluidiccircuit material, the enclosure defining a flow region located withinthe enclosure; and at least one capture structure disposed within theenclosure, where the at least one capture structure includes asolidified polymer network, and wherein the solidified polymer networkincludes an assay reagent and/or assay analyte. In various embodiments,the enclosure of the microfluidic device may include at least onesequestration pen, and the at least one capture structure may bedisposed within the at least one sequestration pen. The at least onesequestration pen may have an isolation region and a connection region,where the connection region may have a proximal opening to the flowregion and a distal opening to the isolation region. In someembodiments, a plurality of capture structures (e.g., 2, 3, 4, etc.) aredisposed within the isolation region of the sequestration pen. Invarious embodiment, the microfluidic device may include a cover.

In another aspect, a method is provided for assaying a micro-object in amicrofluidic device having at least a first in situ-generated capturestructure, the method including: disposing a micro-object within themicrofluidic device in a region proximal to the first in situ-generatedcapture structure, where the in situ-generated capture structureincludes a solidified polymer network, and further where the solidifiedpolymer network includes an assay reagent. The micro-object, such as abiological cell, is allowed to release or produce an analyte; and theanalyte and the assay reagent are allowed to interact. The interactionof the analyte and the assay reagent is detected.

In another aspect, a method is provided for preparing a microfluidicdevice including at least a first in situ-generated capture structure,the method including: providing the microfluidic device, where themicrofluidic device comprises an enclosure including a substrate,microfluidic circuit material, and, optionally, a cover, the enclosuredefining a flow region; introducing a first flowable functionalizedpre-polymer into the flow region; and activating solidification of thefirst flowable functionalized pre-polymer at at least one selected areawithin the enclosure, thereby forming the at least a first insitu-generated capture structure therein. The in situ-generated capturestructure can be formed in the flow region. Alternatively, or inaddition, the enclosure can include at least one sequestration penfluidically connected to the flow region, and the in situ-generatedcapture structure can be formed in the sequestration pen (e.g., anisolation region within the sequestration pen). The step of activatingsolidification of the first flowable functionalized pre-polymer can beperformed at a plurality of selected areas within the enclosure,including within a plurality of sequestration pens and/or at a pluralityof selected areas within each of one or more sequestration pens.

In yet another aspect, a kit is provided, including: a microfluidicdevice having an enclosure including a substrate, microfluidic circuitmaterial, and, optionally, a cover, where the enclosure defines a flowregion; and a functionalized pre-polymer that can be controllablyactivated to form a solidified polymer network. The kit can furtherinclude an assay reagent, which may be part of the functionalizedpre-polymer, mixed with the functionalized pre-polymer, or providedseparately from the functionalized pre-polymer (e.g., in a separatevial, tube, etc.). Alternatively, a kit is provided including: amicrofluidic device having an enclosure including a substrate,microfluidic circuit material, and, optionally, a cover, where theenclosure defines a flow region; and at least one in situ-generatedcapture structure disposed within the enclosure, wherein the at leastone in situ-generated capture structure includes a solidified polymernetwork. The kit can further include an assay reagent, which may beintegral to or associated with the in situ-generated capture structureor which may be provided separately (e.g., in a vial, tube, etc.). Themicrofluidic device in either kit can include at least one sequestrationpen within the enclosure. For kits in which the in situ-generatedcapture structure is already disposed within the microfluidic device,the in situ-generated capture structure can be located within the flowregion, a sequestration pen of the microfluidic device (e.g., anisolation region within the sequestration pen), or both.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates an example of a system for use with a microfluidicdevice and associated control equipment according to some embodiments ofthe disclosure.

FIGS. 1B and 1C illustrate a microfluidic device according to someembodiments of the disclosure.

FIGS. 2A and 2B illustrate isolation pens according to some embodimentsof the disclosure.

FIG. 2C illustrates a detailed sequestration pen according to someembodiments of the disclosure.

FIGS. 2D-F illustrate sequestration pens according to some otherembodiments of the disclosure.

FIG. 2G illustrates a microfluidic device according to an embodiment ofthe disclosure.

FIG. 2H illustrates a coated surface of the microfluidic deviceaccording to an embodiment of the disclosure.

FIG. 3A illustrates a specific example of a system for use with amicrofluidic device and associated control equipment according to someembodiments of the disclosure.

FIG. 3B illustrates an imaging device according to some embodiments ofthe disclosure.

FIGS. 4A-4B are graphical representations of embodiments of insitu-generated assay structures.

FIGS. 4C and 4D are schematic representations of processes forgenerating an assay structure in situ.

FIGS. 5A to 5E are graphical representations of an embodiment of an insitu-generated assay structure of the disclosure, and its use in anassay detecting cytokine secreted by a biological micro-object.

FIGS. 6A-6C are photographic representations of in-situ generated assaystructures detecting low, medium and high secreted amounts of cytokinesecreted by a biological micro-object.

FIG. 7 is a graphical representation of an embodiment of a sequestrationpen including multiple in-situ generated assay structures for multiplexassay of a biological cell.

DETAILED DESCRIPTION OF THE INVENTION

This specification describes exemplary embodiments and applications ofthe invention. The invention, however, is not limited to these exemplaryembodiments and applications or to the manner in which the exemplaryembodiments and applications operate or are described herein. Moreover,the figures may show simplified or partial views, and the dimensions ofelements in the figures may be exaggerated or otherwise not inproportion. In addition, as the terms “on,” “attached to,” “connectedto,” “coupled to,” or similar words are used herein, one element (e.g.,a material, a layer, a substrate, etc.) can be “on,” “attached to,”“connected to,” or “coupled to” another element regardless of whetherthe one element is directly on, attached to, connected to, or coupled tothe other element or there are one or more intervening elements betweenthe one element and the other element. Also, unless the context dictatesotherwise, directions (e.g., above, below, top, bottom, side, up, down,under, over, upper, lower, horizontal, vertical, “x,” “y,” “z,” etc.),if provided, are relative and provided solely by way of example and forease of illustration and discussion and not by way of limitation. Inaddition, where reference is made to a list of elements (e.g., elementsa, b, c), such reference is intended to include any one of the listedelements by itself, any combination of less than all of the listedelements, and/or a combination of all of the listed elements. Sectiondivisions in the specification are for ease of review only and do notlimit any combination of elements discussed.

As used herein, “substantially” means sufficient to work for theintended purpose. The term “substantially” thus allows for minor,insignificant variations from an absolute or perfect state, dimension,measurement, result, or the like such as would be expected by a personof ordinary skill in the field but that do not appreciably affectoverall performance. When used with respect to numerical values orparameters or characteristics that can be expressed as numerical values,“substantially” means within ten percent.

The term “ones” means more than one.

As used herein, the term “plurality” can be 2, 3, 4, 5, 6, 7, 8, 9, 10,or more.

As used herein, the term “disposed” encompasses within its meaning“located.”

As used herein, a “microfluidic device” or “microfluidic apparatus” is adevice that includes one or more discrete microfluidic circuitsconfigured to hold a fluid, each microfluidic circuit comprised offluidically interconnected circuit elements, including but not limitedto region(s), flow path(s), channel(s), chamber(s), and/or pen(s), andat least one port configured to allow the fluid (and, optionally,micro-objects suspended in the fluid) to flow into and/or out of themicrofluidic device. Typically, a microfluidic circuit of a microfluidicdevice will include a flow region, which may include a microfluidicchannel, and at least one chamber, and will hold a volume of fluid ofless than about 1 mL, e.g., less than about 750, 500, 250, 200, 150,100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 μL. In certainembodiments, the microfluidic circuit holds about 1-2, 1-3, 1-4, 1-5,2-5, 2-8, 2-10, 2-12, 2-15, 2-20, 5-20, 5-30, 5-40, 5-50, 10-50, 10-75,10-100, 20-100, 20-150, 20-200, 50-200, 50-250, or 50-300 μL. Themicrofluidic circuit may be configured to have a first end fluidicallyconnected with a first port (e.g., an inlet) in the microfluidic deviceand a second end fluidically connected with a second port (e.g., anoutlet) in the microfluidic device.

As used herein, a “nanofluidic device” or “nanofluidic apparatus” is atype of microfluidic device having a microfluidic circuit that containsat least one circuit element configured to hold a volume of fluid ofless than about 1 μL, e.g., less than about 750, 500, 250, 200, 150,100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nL or less. Ananofluidic device may comprise a plurality of circuit elements (e.g.,at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200,250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000,3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, or more). Incertain embodiments, one or more (e.g., all) of the at least one circuitelements is configured to hold a volume of fluid of about 100 pL to 1nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pL to 5 nL, 250pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15 nL, 750 pL to10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1 to 10 nL, 1 to 15 nL, 1 to 20nL, 1 to 25 nL, or 1 to 50 nL. In other embodiments, one or more (e.g.,all) of the at least one circuit elements are configured to hold avolume of fluid of about 20 nL to 200 nL, 100 to 200 nL, 100 to 300 nL,100 to 400 nL, 100 to 500 nL, 200 to 300 nL, 200 to 400 nL, 200 to 500nL, 200 to 600 nL, 200 to 700 nL, 250 to 400 nL, 250 to 500 nL, 250 to600 nL, or 250 to 750 nL.

A “microfluidic channel” or “flow channel” as used herein refers to flowregion of a microfluidic device having a length that is significantlylonger than both the horizontal and vertical dimensions. For example,the flow channel can be at least 5 times the length of either thehorizontal or vertical dimension, e.g., at least 10 times the length, atleast 25 times the length, at least 100 times the length, at least 200times the length, at least 500 times the length, at least 1,000 timesthe length, at least 5,000 times the length, or longer. In someembodiments, the length of a flow channel is in the range of from about100,000 microns to about 500,000 microns, including any rangetherebetween. In some embodiments, the horizontal dimension is in therange of from about 100 microns to about 1000 microns (e.g., about 150to about 500 microns) and the vertical dimension is in the range of fromabout 25 microns to about 200 microns, e.g., from about 40 to about 150microns. It is noted that a flow channel may have a variety of differentspatial configurations in a microfluidic device, and thus is notrestricted to a perfectly linear element. For example, a flow channelmay be, or include one or more sections having, the followingconfigurations: curve, bend, spiral, incline, decline, fork (e.g.,multiple different flow paths), and any combination thereof. Inaddition, a flow channel may have different cross-sectional areas alongits path, widening and constricting to provide a desired fluid flowtherein. The flow channel may include valves, and the valves may be ofany type known in the art of microfluidics. Examples of microfluidicchannels that include valves are disclosed in U.S. Pat. Nos. 6,408,878and 9,227,200, each of which is herein incorporated by reference in itsentirety.

As used herein, the term “obstruction” refers generally to a bump orsimilar type of structure that is sufficiently large so as to partially(but not completely) impede movement of target micro-objects between twodifferent regions or circuit elements in a microfluidic device. The twodifferent regions/circuit elements can be, for example, the connectionregion and the isolation region of a microfluidic sequestration pen.

As used herein, the term “constriction” refers generally to a narrowingof a width of a circuit element (or an interface between two circuitelements) in a microfluidic device. The constriction can be located, forexample, at the interface between the isolation region and theconnection region of a microfluidic sequestration pen of the instantdisclosure.

As used herein, the term “transparent” refers to a material which allowsvisible light to pass through without substantially altering the lightas is passes through.

As used herein, the term “micro-object” refers generally to anymicroscopic object that may be isolated and/or manipulated in accordancewith the present disclosure. Non-limiting examples of micro-objectsinclude: inanimate micro-objects such as microparticles; microbeads(e.g., polystyrene beads, Luminex™ beads, or the like); magnetic beads;microrods; microwires; quantum dots, and the like; biologicalmicro-objects such as cells; biological organelles; vesicles, orcomplexes; synthetic vesicles; liposomes (e.g., synthetic or derivedfrom membrane preparations); lipid nanorafts, and the like; or acombination of inanimate micro-objects and biological micro-objects(e.g., microbeads attached to cells, liposome-coated micro-beads,liposome-coated magnetic beads, or the like). Beads may includemoieties/molecules covalently or non-covalently attached, such asfluorescent labels, proteins, carbohydrates, antigens, small moleculesignaling moieties, or other chemical/biological species capable of usein an assay. Lipid nanorafts have been described, for example, inRitchie et al. (2009) “Reconstitution of Membrane Proteins inPhospholipid Bilayer Nanodiscs,” Methods Enzymol., 464:211-231.

As used herein, the term “cell” is used interchangeably with the term“biological cell.” Non-limiting examples of biological cells includeeukaryotic cells, plant cells, animal cells, such as mammalian cells,reptilian cells, avian cells, fish cells, or the like, prokaryoticcells, bacterial cells, fungal cells, protozoan cells, or the like,cells dissociated from a tissue, such as muscle, cartilage, fat, skin,liver, lung, neural tissue, and the like, immunological cells, such as Tcells, B cells, natural killer cells, macrophages, and the like, embryos(e.g., zygotes), oocytes, ova, sperm cells, hybridomas, cultured cells,cells from a cell line, cancer cells, infected cells, transfected and/ortransformed cells, reporter cells, and the like. A mammalian cell canbe, for example, from a human, a mouse, a rat, a horse, a goat, a sheep,a cow, a primate, or the like.

A colony of biological cells is “clonal” if all of the living cells inthe colony that are capable of reproducing are daughter cells derivedfrom a single parent cell. In certain embodiments, all the daughtercells in a clonal colony are derived from the single parent cell by nomore than 10 divisions. In other embodiments, all the daughter cells ina clonal colony are derived from the single parent cell by no more than14 divisions. In other embodiments, all the daughter cells in a clonalcolony are derived from the single parent cell by no more than 17divisions. In other embodiments, all the daughter cells in a clonalcolony are derived from the single parent cell by no more than 20divisions. The term “clonal cells” refers to cells of the same clonalcolony.

As used herein, a “colony” of biological cells refers to 2 or more cells(e.g. about 2 to about 20, about 4 to about 40, about 6 to about 60,about 8 to about 80, about 10 to about 100, about 20 about 200, about 40about 400, about 60 about 600, about 80 about 800, about 100 about 1000,or greater than 1000 cells).

As used herein, the term “maintaining (a) cell(s)” refers to providingan environment comprising both fluidic and gaseous components and,optionally a surface, that provides the conditions necessary to keep thecells viable and/or expanding.

As used herein, the term “expanding” when referring to cells, refers toincreasing in cell number.

A “component” of a fluidic medium is any chemical or biochemicalmolecule present in the medium, including solvent molecules, ions, smallmolecules, antibiotics, nucleotides and nucleosides, nucleic acids,amino acids, peptides, proteins, sugars, carbohydrates, lipids, fattyacids, cholesterol, metabolites, or the like.

As used herein, “capture moiety” is a chemical or biological species,functionality, or motif that provides a recognition site for amicro-object. A selected class of micro-objects may recognize the insitu-generated capture moiety and may bind or have an affinity for thein situ-generated capture moiety. Non-limiting examples includeantigens, antibodies, and cell surface binding motifs.

As used herein, “flowable polymer” is a polymer monomer or macromer thatis soluble or dispersible within a fluidic medium (e.g., a pre-polymersolution). The flowable polymer may be input into a microfluidic flowregion and flow with other components of a fluidic medium therein.

As used herein, “photoinitiated polymer” refers to a polymer (or amonomeric molecule that can be used to generate the polymer) that uponexposure to light, is capable of crosslinking covalently, formingspecific covalent bonds, changing regiochemistry around a rigidifiedchemical motif, or forming ion pairs which cause a change in physicalstate, and thereby forming a polymer network. In some instances, aphotoinitiated polymer may include a polymer segment bound to one ormore chemical moieties capable of crosslinking covalently, formingspecific covalent bonds, changing regiochemistry around a rigidifiedchemical motif, or forming ion pairs which cause a change in physicalstate. In some instances, a photoinitiated polymer may require aphotoactivatable radical initiator to initiate formation of the polymernetwork (e.g., via polymerization of the polymer).

As used herein, “antibody” refers to an immunoglobulin (Ig) and includesboth polyclonal and monoclonal antibodies; primatized (e.g., humanized);murine; mouse-human; mouse-primate; and chimeric; and may be an intactmolecule, a fragment thereof (such as scFv, Fv, Fd, Fab, Fab′ andF(ab)′2 fragments), or multimers or aggregates of intact moleculesand/or fragments; and may occur in nature or be produced, e.g., byimmunization, synthesis or genetic engineering. An “antibody fragment,”as used herein, refers to fragments, derived from or related to anantibody, which bind antigen and which in some embodiments may bederivatized to exhibit structural features that facilitate clearance anduptake, e.g., by the incorporation of galactose residues. This includes,e.g., F(ab), F(ab)′2, scFv, light chain variable region (VL), heavychain variable region (VH), and combinations thereof.

As used herein in reference to a fluidic medium, “diffuse” and“diffusion” refer to thermodynamic movement of a component of thefluidic medium down a concentration gradient.

The phrase “flow of a medium” means bulk movement of a fluidic mediumprimarily due to any mechanism other than diffusion. For example, flowof a medium can involve movement of the fluidic medium from one point toanother point due to a pressure differential between the points. Suchflow can include a continuous, pulsed, periodic, random, intermittent,or reciprocating flow of the liquid, or any combination thereof. Whenone fluidic medium flows into another fluidic medium, turbulence andmixing of the media can result.

The phrase “substantially no flow” refers to a rate of flow of a fluidicmedium that, averaged over time, is less than the rate of diffusion ofcomponents of a material (e.g., an analyte of interest) into or withinthe fluidic medium. The rate of diffusion of components of such amaterial can depend on, for example, temperature, the size of thecomponents, and the strength of interactions between the components andthe fluidic medium.

As used herein in reference to different regions within a microfluidicdevice, the phrase “fluidically connected” means that, when thedifferent regions are substantially filled with fluid, such as fluidicmedia, the fluid in each of the regions is connected so as to form asingle body of fluid. This does not mean that the fluids (or fluidicmedia) in the different regions are necessarily identical incomposition. Rather, the fluids in different fluidically connectedregions of a microfluidic device can have different compositions (e.g.,different concentrations of solutes, such as proteins, carbohydrates,ions, or other molecules) which are in flux as solutes move down theirrespective concentration gradients and/or fluids flow through themicrofluidic device.

As used herein, a “flow path” refers to one or more fluidicallyconnected circuit elements (e.g. channel(s), region(s), chamber(s) andthe like) that define, and are subject to, the trajectory of a flow ofmedium. A flow path is thus an example of a swept region of amicrofluidic device. Other circuit elements (e.g., unswept regions) maybe fluidically connected with the circuit elements that comprise theflow path without being subject to the flow of medium in the flow path.

As used herein, “isolating a micro-object” confines a micro-object to adefined area within the microfluidic device. The micro-object may stillbe capable of motion within an in situ-generated capture structure.

A microfluidic (or nanofluidic) device can comprise “swept” regions and“unswept” regions. As used herein, a “swept” region is comprised of oneor more fluidically interconnected circuit elements of a microfluidiccircuit, each of which experiences a flow of medium when fluid isflowing through the microfluidic circuit. The circuit elements of aswept region can include, for example, regions, channels, and all orparts of chambers. As used herein, an “unswept” region is comprised ofone or more fluidically interconnected circuit element of a microfluidiccircuit, each of which experiences substantially no flux of fluid whenfluid is flowing through the microfluidic circuit. An unswept region canbe fluidically connected to a swept region, provided the fluidicconnections are structured to enable diffusion but substantially no flowof media between the swept region and the unswept region. Themicrofluidic device can thus be structured to substantially isolate anunswept region from a flow of medium in a swept region, while enablingsubstantially only diffusive fluidic communication between the sweptregion and the unswept region. For example, a flow channel of amicro-fluidic device is an example of a swept region while an isolationregion (described in further detail below) of a microfluidic device isan example of an unswept region.

The capability of biological micro-objects (e.g., biological cells) toproduce specific biological materials (e.g., proteins, such asantibodies) can be assayed in such a microfluidic device. In a specificembodiment of an assay, sample material comprising biologicalmicro-objects (e.g., cells) to be assayed for production of an analyteof interest can be loaded into a swept region of the microfluidicdevice. Ones of the biological micro-objects (e.g., mammalian cells,such as human cells) can be selected for particular characteristics anddisposed in unswept regions. The remaining sample material can then beflowed out of the swept region and an assay material flowed into theswept region. Because the selected biological micro-objects are inunswept regions, the selected biological micro-objects are notsubstantially affected by the flowing out of the remaining samplematerial or the flowing in of the assay material. The selectedbiological micro-objects can be allowed to produce the analyte ofinterest, which can diffuse from the unswept regions into the sweptregion, where the analyte of interest can react with the assay materialto produce localized detectable reactions, each of which can becorrelated to a particular unswept region. Any unswept region associatedwith a detected reaction can be analyzed to determine which, if any, ofthe biological micro-objects in the unswept region are sufficientproducers of the analyte of interest.

Microfluidic Devices with In Situ-Generated Capture Structures.

It can be advantageous when performing assays upon a micro-object withina microfluidic device that such assays may incorporate an assay analyteor assay reagent that is affixed (e.g. by adhering the assay analyte orassay reagent, or limiting the motion and/or diffusion of the assayanalyte or assay reagent) to a specific area and/or feature of themicrofluidic circuit, such as a sequestration pen, a trap, or a portionof a flow region, including but not limited to a microfluidic channel.In some instances, the assay analyte or assay reagent may be affixed toa specific portion of the microfluidic device (e.g., a portion of asequestration pen) using a polymer network. The solidified polymernetwork may be generated in situ at a selected location. For example,structured light may be used to generate a solidified network ofpolymers through a light-induced polymerization/cross-linking reactionthat solidifies the polymers by cross linking the polymers into anetwork. The solidified polymer network can be reacted with (eitherduring or after formation of the solidified polymer network) an assayreagent or assay analyte, thereby forming an in situ-generated capturestructure comprising the assay reagent or assay analyte. The assayreagent or assay analyte can, in this manner, be maintained within, orat least in close proximity to the surface of, the solidified polymernetwork, thereby optimizing the assay (e.g., by concentrating the assaysignal in one or more pre-defined locations).

It has been surprisingly discovered that a wide variety of capturestructures can be generated in-situ within a microfluidic (ornanofluidic) device as described herein. Microfluidic devices,compositions and methods of use for these classes of devices having insitu-generated capture structures are described herein.

A microfluidic device 400 may be provided, including an enclosurecomprising a substrate, and microfluidic circuit material 260, theenclosure defining a flow region (e.g., flow channel 410) and at leastone sequestration pen 430, each located within the enclosure (notshown); and at least one in situ-generated capture structure 404disposed within the enclosure, wherein the at least one capturestructure 404 comprises a solidified polymer network. The microfluidiccircuit material 260 may define the walls of the flow region, and maydefine other microfluidic circuit elements within the enclosure. In someembodiments, the microfluidic device 400 may include a cover (notshown). In various embodiments, the microfluidic device may include atleast one sequestration pen 430, which may also be formed ofmicrofluidic circuit material 260. In some embodiments, the at least onein situ-generated capture structure 404 may be disposed within the atleast one sequestration pen 430. The microfluidic device may furtherinclude a plurality of sequestration pens within the enclosure. The atleast one in situ-generated capture structure is configured to becapable of capturing a biological product of a micro-object and/or beacted upon by the micro-object or a biological product of themicro-object. The at least one in situ-generated capture structure mayinclude an assay reagent or assay analyte, or may be configured toaccept an assay reagent or assay analyte (e.g., may includefunctionalized sites configured to react with a functionalized assayreagent or assay analyte). An assay reagent may be configured to capturea biological product of the micro-object. An assay analyte may beconfigured to capture a biological product of a micro-object or to beacted upon by a micro-object or a biological product of themicro-object.

A portion of microfluidic device 400 is shown in FIG. 4A. The at leastone sequestration pen 430 may be fluidically connected to the flowregion (e.g., flow channel 410). The at least one sequestration pen 430may include an isolation region and a connection region, and have anyset of dimensions as described above for any sequestration pen 124, 126,128, 130, 224, 226, 228, 266 where the connection region has a proximalopening to the flow region (e.g., flow channel 410) and a distal openingto the isolation region. The flow region (e.g., flow channel 410) of themicrofluidic device may include a microfluidic channel 410. The proximalopening of the sequestration pen to the flow region (e.g., flow channel410) may be oriented substantially parallel to a flow of fluidic mediumin the flow region (not shown). Exchange of components of betweenfluidic media in the flow region and fluidic media within the isolationregion of the sequestration pen may occur substantially only bydiffusion. The at least one in situ-generated capture structure 404 maybe disposed within the isolation region of the sequestration pen 430.

The at least one in situ-generated capture structure 404 may be locatedwithin a connection region or an isolation region of a sequestrationpen. The in situ-generated capture structure 404 may further beselectively formed to be in a location of the isolation region of thesequestration pen such that cells may be imported into the isolationregion without hindrance. The at least one in situ-generated capturestructure 404 may be located within the isolation region such that cellsmay be exported out of the isolation region without hindrance from thein situ-generated capture structure 404. The microfluidic device mayinclude a plurality of sequestration pens 430, which may be configuredin any suitable arrangement as described herein, in any combination.When a microfluidic channel and a plurality of sequestration pens arepresent, the plurality of sequestration pens may be aligned in a row,with each sequestration pen of the plurality opening off of one side ofthe microfluidic channel 410. The proximal openings of eachsequestration pen of the plurality may open to the microfluidic channel410 in a common direction.

In another embodiment, a microfluidic device 450 is provided, a portionof which is as shown in FIG. 4B, including an enclosure comprising asubstrate and a cover, the enclosure defining a flow region (e.g., flowchannel 410) and at least one sequestration pen 430, each located withinthe enclosure (not shown); and at least one in situ-generated capturestructure 406 disposed within the at least one sequestration pen 430,wherein the at least one in situ-generated capture structure 406comprises a solidified polymer network which further comprises an assayreagent or assay analyte (R/A, e.g., 406B or 406A of FIGS. 4C and 4D).

Substrate.

The substrate of the microfluidic device 400, 450 may further include aconfiguration for generating dielectrophoresis (DEP) forces within theenclosure (not shown). The microfluidic device substrate having a DEPconfiguration may include any DEP configuration as described herein. TheDEP forces may be optically actuated. In other embodiments, thesubstrate of the microfluidic device 400, 450 may be configured toinclude an opto-electrowetting configuration (not shown). In someembodiments, the opto-electrowetting substrate may be opticallyactuated. In yet other embodiments, the microfluidic device 400, 450 mayinclude a combination of a substrate configured to generate DEP forcesand a substrate configured to generate electrowetting forces, each ofwhich are optically actuated.

In some embodiments, the cover of the microfluidic device 400, 450 maybe substantially transparent to a fluorescent, colorimetric, orluminescent signal from the one or more in situ-generated capturestructures.

In various embodiments, the microfluidic device having at least onecapture structure 400, 450 may have a dynamic coating or a conditionedsurface which enhances cell growth, viability, portability and anycombination thereof, as described above. Any suitable dynamic coating orconditioned surface may be used. In some embodiments, a conditionedsurface may include a covalently modified surface, which may be anysuitable covalently modified surface as described herein. A covalentlymodified surface may be present before solidifying the polymer networkof the in situ-generated capture structure. If a dynamic coating isused, it may be introduced before or after solidifying the polymernetwork of the in situ-generated capture structure.

Microfluidic device 400, 450 may have any other components, features orconfigurations as described for microfluidic devices 100, 200, 230, 250,280, 290, 320, 500, 700 described herein, in any combination.

In Situ-Generated Capture Structure Including a Solidified PolymerNetwork.

The solidified polymer network of the in situ-generated capturestructures 404, 406 (FIGS. 4A, 4B) may include a photoinitiated polymer,and may be solidified in situ. In some embodiments, the solidifiedpolymer network does not include a silicone polymer. In someembodiments, the solidified polymer network does not include silicon.The solidified polymer network may be made from any suitable polymer andmay be any polymer as described herein.

Functionalized Sites.

The solidified polymer network of the at least one in situ-generatedcapture structure 404 of microfluidic device 400 may include one or morefunctionalized sites. In some embodiments, the solidified polymernetwork of the in situ-generated capture structure may include two ofmore functionalized sites. The functionalized sites may be adhered(which may include non-specific non-covalent binding or may includenon-covalent binding via a specific binding pair or motif) to thesolidified polymer network. In other embodiments, the functionalizedsites of the solidified polymer network may be covalently bound to thepolymer(s) of the solidified polymer network.

The functionalized sites may include a reactive moiety R_(fs) permittingan assay reagent or assay analyte to be introduced thereto. The reactivemoiety R_(fs) may provide a covalent or non-covalent mode of reaction,including association (e.g., chelation, for one non-limiting example),binding (e.g., non-covalent binding such as between biotin andstreptavidin or an antibody/antigen binding pair), or reaction (e.g.,forming a covalent bond such as between Click reaction pairs). Forsimplicity, the term binding may be used to encompass all three types ofinteractions, but one or more of these interactions may be preferred inspecific embodiments. In some embodiments, the reactive moiety R_(fs) ofthe one or more functionalized sites may be biotin, avidin orstreptavidin. In other embodiments, the reactive moiety R_(fs) of theone or more functionalized sites may include a chelating moiety or anoligonucleotide hybridization sequence. In some embodiments, the one ormore functionalized sites of the solidified polymer network may beintroduced after solidification of the polymer network (e.g., anon-specifically adhered species containing a reactive moiety R_(fs) maybe flowed into the sequestration pen and permitted to contact thesolidified polymer network for a period of time to adhere sufficientnumbers of the species containing reactive moiety R_(fs) for suitableassay conditions) as shown schematically for the conversion of insitu-generated capture structure 403 including a solidified polymernetwork, to in situ-generated capture structure 404 including asolidified polymer network having at least one functionalized site. Inother embodiments, the one or more functionalized sites including areactive moiety R_(fs) are introduced to the prepolymer 401 prior tosolidification of the polymer network, as shown schematically in FIGS.4C and 4D.

Microfluidic device 450, having at least in situ-generated one capturestructure 406 which includes an assay reagent or assay analyte (e.g.,R/A of 406 of FIG. 4B), may contain one or more functionalized siteseach having a reactive moiety R_(fs) already associated, bound orreacted with an assay reagent or assay analyte. The reactive moietyR_(fs) may be selected from any reactive moiety as described above formicrofluidic device 400 and respective in situ-generated capturestructure 404. As above, the term binding may be used to encompass allthree types of interactions, but one or more of these interactions maybe preferred in specific embodiments. Binding of the assay reagent orassay analyte may be conducted either prior to solidifying the polymernetwork or subsequent to solidification, as described below and shown inFIG. 4C.

In some embodiments, the one or more functionalized sites of asolidified polymer network of an in situ-generated capture structure404, 406 may all include the same reactive moiety R_(fs). In otherembodiments, the one or more functionalized sites of a solidifiedpolymer network of an in situ-generated capture structure 404, 406 mayinclude different R_(fs). In some other embodiments, more than one typeof polymer may be used to form the solidified polymer network and eachpolymer may have the same or different functionalized sites (e.g.,reactive moieties R_(fs) attached or adhered thereto).

Assay Reagent or Assay Analyte.

The solidified polymer network of the at least one in situ-generatedcapture structure 406 may further include an assay reagent and/or assayanalyte (FIGS. 4B, 4C, 4D). The in situ-generated capture structure 406of the microfluidic device 450 may be provided already including anassay reagent or an assay analyte bound to the solidified polymernetwork. Alternatively, the microfluidic device 400 may have an insitu-generated capture structure 404 (FIG. 4A) configured to associate,bind or react with the assay reagent or assay analyte to provide an insitu-generated capture structure 406 including an assay reagent or assayanalyte (R/A of FIG. 4B), and shown in more schematic detail in FIG. 4C.In yet another alternative, the solidified polymer network and itsassociated assay reagent or assay analyte may be introduced before thestart of the assay experiment itself. The assay reagent or assay analytemay be configured to be covalently or non-covalently bound to the one ormore functionalized sites of the solidified polymer network. The assayreagent or assay analyte may be introduced during the initial formationof the in situ-generated capture structure, for example, by beingcovalently bound to the flowable polymer solution (e.g., alreadyincorporated within the pre-polymer). One non-limiting example may beincorporation of recognition motifs such as an RGD motif, which may berecognized by integrins on a target biological cell.

Alternatively, the assay reagent or assay analyte may be flowed into themicrofluidic device 400 having an in situ-generated capture structure404 including one or more functionalized sites (e.g., at some time afterthe solidified polymer network has been solidified). The assay reagentor assay analyte may include a functional moiety M_(fs) configured toassociate, bind or react with R_(fs) of the functionalized sites of thesolidified polymer network of the at least one in situ-generated capturestructure 404 to generate the at least one in situ-generated capturestructure 406. As above, the term binding may be used to encompass allthree types of interactions, but one or more of these interactions maybe preferred in specific embodiments. Any suitable functional moietyM_(fs) may be used. For example, a chelating substrate M_(fs) of anassay reagent or assay analyte may be chelated by a chelating ligandR_(fs) of the functionalized sites of the solidified polymer network ofcapture structure 404. A functional moiety M_(fs) may include biotin orstreptavidin, to bind non-covalently with a respective avidin,streptavidin or biotin R_(fs) of the functionalized sites of capturestructure 404. Alternatively, a functional moiety M_(fs) may beconfigured to react covalently with the R_(fs) of the functionalizedsites of the solidified polymer network. For example, a functionalmoiety M_(fs) may be an azide and may react covalently with an alkynylfunctionality of a corresponding Click reaction pair of a functionalizedsite of an in situ-generated capture structure 404.

In some embodiments of the microfluidic device including at least one insitu-generated capture structure, the assay reagent or assay analyte mayinclude a detectable label. The detectable label of the assay reagent orassay analyte may be a fluorescent, colorimetric, or luminescent label.In some embodiments, the detectable label may be a fluorescent label. Insome embodiments, when the assay reagent or assay analyte includes adetectable label, the label is not detectable until the assay isproceeding, and the detectable label is generated or liberated from theassay reagent or assay analyte.

Methods of Introducing Solidified Polymer Networks which May Include aReactive Moiety and/or Assay Reagent or Assay Analyte.

Preparation of the solidified polymer network of the at least one insitu-generated capture structure may be performed variously, as shownschematically in FIGS. 4C and 4D. In one route shown in FIG. 4C, one ormore prepolymers 401 may be modified to provide a prepolymer 405containing at least one functionalized site including a reactive moietyR_(fs). This unsolidified prepolymer 405 may subsequently be flowed intothe enclosure of the microfluidic device, and solidified in-situ toprovide an in situ-generated capture structure 404, which may optionallyinclude introducing the flowable pre-polymer into a sequestration pen.Alternatively, the prepolymer 405 having at least one functionalizedsite including a reactive moiety R_(fs), may be reacted with an assayanalyte having a functional moiety M_(fs) to provide a prepolymer 407Aalready containing an assay analyte. This prepolymer 407A, alreadyincorporating an assay analyte, may subsequently be flowed into themicrofluidic device, and optionally to the sequestration pen, and may besolidified in situ to provide an in situ-generated capture structure406A having an assay analyte.

In another embodiment, the prepolymer 405 having at least one functionalsite including a reactive moiety R_(fs) is reacted with an assay reagenthaving a functional moiety M_(fs) to provide a prepolymer 407B alreadycontaining an assay reagent. The prepolymer 407B may subsequently beflowed into the enclosure of the microfluidic device, and optionallyintroduced to the sequestration pen, and solidified in situ to providean in situ-generated capture structure 406B including an assay reagent.

In yet another embodiment, prepolymer 401 itself may be flowed into theenclosure of the microfluidic device, and optionally to thesequestration pen, and may be solidified in situ to provide thesolidified polymer network 403 forming part of the in situ-generatedcapture structure. The solidified polymer network may be modified tointroduce at least one functional site having a reactive group R_(fs) byflowing in a material to adhere or bond to the solidified polymernetwork, thereby providing a solidified polymer network including atleast one reactive group R_(fs) (e.g., in situ-generated capturestructure 404). In situ-generated capture structure 404 may be furthermodified by flowing in an assay analyte having a functional moietyM_(fs), which reacts with the R_(fs) of capture structure 404 to providean in situ-generated capture structure 406A containing an assay analyte.Alternatively, in situ-generated capture structure 404 may be furthermodified by flowing in an assay reagent having a functional moietyM_(fs), which reacts with the R_(fs) of capture structure 404 to providean in situ-generated capture structure 406B containing an assay reagent.

In yet another embodiment, shown in FIG. 4D, prepolymer 401′ is preparedhaving an assay analyte or assay reagent already incorporated into theprepolymer, such as, for example, a peptide segment including an RGD ora proteinase substrate (e.g., PEP in FIG. 4D) motif. Prepolymer 401′ maybe flowed into the enclosure of the microfluidic device, and optionallyto the sequestration pen, and solidified in situ to provide an insitu-generated capture structure 406C, having an assay reagent or assayanalyte incorporated within the solidified polymer network.

Methods of introducing the in situ-generated capture structures aredescribed in fuller detail below.

Assay Reagent of the In Situ-Generated Capture Structure.

The assay reagent may include a protein, a nucleic acid, an organicmolecule, and/or a saccharide. The assay reagent may include an insitu-generated capture oligonucleotide, which can hybridize to a nucleicacid of interest. The oligonucleotide may be synthetically produced ormay be produced by a biological cell. The oligonucleotide may be furtherprocessed after biological production for size or to introduce otherfunctionality. A protein assay reagent may include, but is not limitedto an antibody, a structural protein, a cell surface marker, or acytokine. An organic molecule assay reagent may include a synthetic,semi-synthetic or biologically produced organic molecule having amolecular weight of about 2000 Da or less. An organic molecule mayinclude a chelation substrate, a chelation ligand, a peptide, or anon-peptidic organic molecule. In some embodiments, the assay reagentmay include a combination of two or more of a protein, nucleic acid, anorganic molecule, or a saccharide. In some embodiments, the assayreagent may include an antibody or a fragment thereof. In otherembodiments, the assay reagent may include an antigen. In someembodiments, the antigen assay reagent may be a cytokine, including butnot limited to tumor necrosis factor alpha (TNF alpha), interferon alpha(IFN-alpha), Interleukin 2 (IL-2) or IFN gamma.

In some embodiments, when the assay reagent includes an antibody, theassay reagent antibody may specifically bind to a tumor antigen, whichmay be any tumor antigen as described herein. In other embodiments, theassay reagent antibody may specifically bind to a cytokine, which may beany suitable cytokine, including but not limited to tumor necrosisfactor alpha (TNF alpha), interferon alpha (IFN-alpha), Interleukin 2(IL-2) or IFN gamma.

In some embodiments, when the assay reagent includes an antigen, theantigen reagent may be a tumor antigen. The tumor antigen reagent may bea tumor specific antigen or a tumor associated antigen. A non-limitinglist of tumor antigens that may be used as an assay reagent include WT1,MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2/neu, MAGE A3, p53 nonmutant,NY-ESO-1, PSMA, GD2, CEA, MelanA/MART1, Ras mutant, gp100, p53 mutant,Proteinase 3 (PR1), bcr-able. Tyrosinase, Survivin, PSA, hTERT, EphA2,PEP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene, NA17, PAX3, ALK,Androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD3,fucosyl GM1, Mesothelin, or PSCA.

FIG. 5A shows one example of a microfluidic device 500, having at leastone sequestration pen 530 opening to microfluidic channel 264. Thesequestration pen 530 has one in situ generated capture structure 502having a solidified polymer network which includes an assay reagent 504,shown here as an antibody.

These examples of an assay reagent are in no way limiting, and may beany suitable assay reagent as one of skill may select.

Assay Analyte of the In Situ-Generated Capture Structure.

An assay analyte may bind to the solidified polymer network of the insitu-generated capture structure via a covalent or non-covalent bindinginteraction with the assay reagent. In some of the embodiments when anassay analyte is bound to/incorporated within the in situ-generatedcapture structure, the assay analyte also includes a detectable labelsuch as a fluorescent, luminescent or visibly colored dye label. Theassay analyte may include a protein, a nucleic acid, an organic molecule(as described above), and/or a saccharide. In some embodiments, theassay analyte may include a combination of two or more of a protein,nucleic acid, an organic molecule, or a saccharide. A protein assayanalyte may include, but is not limited to an antibody, a structuralprotein, a cell surface marker, or a cytokine.

In some embodiments, the assay analyte may include an antibody or afragment thereof. In one non-limiting example, it is not uncommon whenstudying antibodies to screen for an “anti-idiotype” antibody that bindsthe binding site of a first antibody. The anti-idiotype antibody canmimic the antigen bound by the first antibody, and thereby can be usedto (1) model the antigen bound by the first antibody, or (2) vaccinatean animal (thus creating new antibodies that are similar to the firstantibody. The anti-idiotype antibody can therefore be viewed as an assayanalyte in this context, or may alternatively be considered an assayreagent and used accordingly.

In other embodiments, the protein assay analyte may be an antigennon-covalently bound to the in situ-generated capture structure. Anorganic molecule assay analyte may include a peptide, or a non-peptidicorganic molecule. One non-limiting example of an assay analyte is asubstrate for a proteinase. The substrate may be a peptidic or anon-peptidic organic molecule. The assay may identify a cell thateffectively produces a proteinase of interest, which may be of use forcommercial production. Alternatively, the substrate may be the target ofa pathogenic proteinase expression and can be used to identify cellshaving the pathogenic activity.

For example, a matrix metalloproteinase (MMP) substrate (such as, forexample, MMP-2, which may have a substrate sequence ofGly-Pro-Gln-Gly-Trp-Gly-Gln, (e.g., PEP)) may be incorporated into an insitu-generated capture structure by any suitable manner such asincorporation within the pre-polymer (e.g, 401′) or introduced into afunctionalized site of an in situ-generated capture structure (e.g., viaa crosslinker, yielding a prepolymer 407A, and/or in situ-generatedcapture structure 406A). Expression of certain metalloproteinases isassociated with metastatic potential and cancer progression. An insitu-generated capture structure incorporating an MMP substrate motifmay be used to identify cells expressing the MMP. If the MMP substrateis part of the solidified polymer network of the in situ-generatedcapture structure 406C (See FIG. 4D), the solidified polymer network maybe eroded and loss of the network may be monitored. For example, thesolidified polymer network incorporating a MMP substrate motif mayfurther include a fluorescent label that is liberated as the proteinaseactivity continues. Loss of signal within the solidified polymer networkmay be monitored or gain of signal within the liquid medium within thesequestration pen may be monitored. Substrates that may be bound orincorporated within the solidified polymer network of the insitu-generated capture structure are not limited to any specific type ofsubstrates but may be any suitable substrate for which assay may bedesired.

Another protease substrate that may be a useful assay analyte to bebound or incorporated within an in situ-generated capture structure maybe a furin substrate. Furin (proprotein convertase having serineendoprotease activity) may be involved in differentiation of T cells toa Th1 phenotype. The assay may be performed in various ways using the insitu-generated capture structure as described herein, but in oneembodiment, a peptide may incorporate a cleavage motif for Furin, afunctional moiety M_(fs), such as biotin, which permits attachment tofunctional sites within the solidified polymer network of the insitu-generated capture structure, and a fluorophore attached to alocation within the peptide which will be released upon cleavage byfurin. Cells expressing furin activity would release fluorescence fromthe in situ-generated capture structure, and the loss of signal may bedetected and may further be quantitated.

In yet another embodiment, a fluorescently labeled antigen may beembedded, either by adhesion or possibly by another non-covalent mode,within the solidified polymer network of the in situ-generated capturestructure 406. An assay may be performed to measure antigen extractionby B cells. As the B cells associate or bind with the solidified polymernetwork, if the B cell expresses an antibody specific for the antigen,the antigen may be extracted from the solidified polymer network. Higheraffinity antibodies may exhibit higher levels of antigen extraction, andhence loss of fluorescent signal from the solidified polymer network ofthe in situ-generated capture structure.

These examples of an assay analyte are in no way limiting, and the assayanalyte may be any suitable assay analyte as one of skill may select

Detection Reagent(s).

The result of the interaction between the assay reagent (or analyte) andits intended target may be detected by a detection reagent. Thedetection reagent may include a detectable label. The detectable labelof the detection reagent may include a fluorescent, colorimetric, orluminescent label. In some embodiments, the detection reagent mayinclude at least a first antibody. A detection reagent may include afirst antibody that is detectably labelled. In some embodiments, thedetection reagent may include a second antibody, where the secondantibody may incorporate the detectable label. In some embodiments,where the labeled second antibody is a secondary antibody and binds tothe at least first antibody. The first and/or the second antibody may bean IgG antibody. The first and/or the second antibody may be a fragmentof an antibody. In other embodiments, the detection reagent may includean intercalating dye. In yet other embodiments, the detection reagentmay include a FRET labeled oligonucleotide, which may include but is notlimited to a molecular beacon, dual hybridization probe, Scorpion®, orEclipse® probe. A FRET labeled oligonucleotide probe or probe pair mayinclude fluorescent labels that do not fluoresce until a hybridizationevent takes place. The detection reagent may be an intercalating dye,including but not limited to phenanthridine or acridine dyes.

Microfluidic Devices Having One or More In Situ-Generated CaptureStructures for Multiplexed Assays.

The at least one in situ-generated capture structure in the enclosure,and optionally where the at least one in situ-generated capturestructure may be disposed within at least one sequestration pen, of themicrofluidic device 400, 450, may be configured to detect more than oneinteraction (e.g., may have two, three or more different assay reagentsor analytes bound to the in situ-generated capture structure), thusproviding one mode of conducting multiplexed assays. In someembodiments, a single in situ-generated capture structure, of theenclosure or the at least one sequestration pen, is configured tocontain two assay reagents that detect two different analytes (e.g.,biological products of a cell).

In other embodiments, the enclosure of the microfluidic device 400, 450may include two or more in situ-generated capture structures disposedtherein. In some embodiments, the at least one sequestration pen of themicrofluidic device 400, 450 may include the two or more insitu-generated capture structures disposed therein. The two or more insitu-generated capture structures may be disposed within the isolationregion of the sequestration pen. For a microfluidic device 450, a firstsolidified polymer network of a first in situ-generated capturestructure may include a first assay reagent or assay analyte and asecond solidified polymer network of a second in situ-generated capturestructure may include a second assay reagent or assay analyte, and so onfor each additional in situ-generated capture structure in the at leastone sequestration pen. For microfluidic device 400, a first solidifiedpolymer network of a first in situ-generated capture structure mayinclude a first type of functionalized sites and a second solidifiedpolymer network of a second in situ-generated capture structure mayinclude a second type of functionalized sites, which can each accept adifferent kind of assay reagent or assay analyte. In the embodiments ofmicrofluidic devices 400, 450, having multiple in situ-generated capturestructures within the enclosure or within the at least one sequestrationpen, the first assay reagent or assay analyte may be different from thesecond assay reagent or assay analyte, and so on for each additionalassay reagent or assay analyte. The first in situ-generated capturestructure and the second in situ-generated capture structure may bedisposed in different locations within the enclosure or, alternatively,within at least one sequestration pen of the microfluidic device. Thefirst in situ-generated capture structure and the in situ-generatedsecond capture structure may be disposed on a first wall and a secondwall, respectively of the enclosure or may be located adjacent to oneanother on the same wall. The first in situ-generated capture structureand the in situ-generated second capture structure may be disposed on afirst wall and a second wall, respectively of the sequestration pen, ormay be disposed adjacent to each other on a first wall of thesequestration pen.

An example of a microfluidic device including at least one sequestrationpen, where the sequestration pen includes more than one insitu-generated capture structure is shown in FIG. 7. One portion ofmicrofluidic device 700 is shown, displaying one sequestration pen 730.Microfluidic device 700 may have any combination of components andfeatures of any of the microfluidic devices 100, 200, 230. 250, 280,290, 400, 450, 500, in any suitable combination as may be selected byone of skill. The sequestration pen 730 may be constructed of the samemicrofluidic circuit material 260, which also defines channel 264. Afirst fluidic medium (not shown) may flow with flow 278 in themicrofluidic channel 264. Sequestration pen 730 has three capturestructures 702, 704, 708 disposed in three physically distinguishablelocations within the pen 730. There are two micro-objects 706 loadedinto the pen 730, which, in this embodiment, are producing biologicalproducts 716, 718, and 720. As few as one micro-object 706 may bepresent, or there may be a plurality of micro-objects 706, as may besuitable for a selected assay. The biological products 716, 718, 720 maybe all different or may be the same biological product which is assayedfor three different characteristics by the assay reagents 710, 712, 714(which alternatively can be any selection of assay reagent and/or assayanalyte) which are included respectively within and/or on capturestructures 702, 704, 708, forming in situ capture structures (702 plus710), (704 plus 712), and (708 plus 714), which are equivalent to insitu-generated capture structure 406 of FIG. 4B, or in situ-generatedcapture structures 406A and/or 406B of FIG. 4C. The assay reagents 710,712, 714 are each different from each other and test for either adifferent biological product or a different characteristic of abiological product. As shown in FIG. 7, the assay reagents 710, 712, 714are shown as antibodies for ease of viewing, but the multiplexed insitu-generated capture structures are not limited to including onlyantibody assay reagents, but may be any suitable combination of assayreagents (or assay analytes) as described herein.

Other Properties of the at Least One Capture Structure.

When the in situ-generated capture structure is located within theenclosure, and optionally within the flow region, a size of the insitu-generated capture structure may have any suitable size that maypermit flow of the fluidic medium through the flow region. In someembodiments, the in situ-generated capture structure may have adimension across the flow region (which may be a microfluidic channel)that is less than 80%, 70%, 60%, 50% 40%, 30% 20%, 10%, 5%, 1%, or lessof a width of the flow region. The isolation region of the sequestrationpen of the microfluidic device may have a width of about 50 microns toabout 250 microns, and a width of the in situ-generated capturestructure generated therein may be in a range from about ⅛ to about ¾ ofthe width of the isolation region, or any value therebetween. A width ofthe in situ-generated capture structure across the isolation region maybe in a range of about 5 microns to about 35 microns (or any valuetherebetween) in an isolation region having a width of about 50 micronsor in a range of about 60 microns to about 190 microns (or any valuetherebetween) in an isolation region having a width of about 250microns. In various embodiments, the in situ-generated capture structuremay be configured to permit exit of a micro-object, including but notlimited to a biological micro-object (e.g., a biological cell or embryo)or microbead, from the sequestration pen.

In some embodiments, the in situ-generated capture structure may beporous to a flow of fluidic medium. The solidified polymer network maynot be porous to at least a subset of a plurality of micro-objects. Insome embodiments, the solidified polymer network is substantiallynon-porous to micro-object having a diameter of greater than about 1 nm,2 nm, 10 nm, 100 nm, 250 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1micron, 2 microns, 3 microns, 4 microns, 5 microns, 6 microns, 7microns, 8 microns, 9 microns, 10 microns, 11 microns, 12 microns, 13microns, 14 microns, 15 microns, or more.

In some embodiments, at least a portion of the in situ-generated capturestructure may be removable. The in situ-generated capture structure maybe at least partially removable by hydrolysis, proteolysis, osmoticchange, temperature change, or optical illumination, as discussed below.

Other Features of the Microfluidic Device Having at Least One InSitu-Generated Capture Structure.

The microfluidic device may be any microfluidic device described hereinand may include any components, features or dimensions described belowin any combination.

In some embodiments, the enclosure of the microfluidic device mayfurther include a selection sector. The selection region may contain theat least one in situ-generated capture structure and at least part ofthe flow region. The selection sector may be a distinct region of theenclosure of the microfluidic device where assays are performed asdescribed herein.

In some embodiments, the enclosure of the microfluidic device mayfurther include an isolation sector. The isolation sector may be used tomaintain, grow and/or expand selected micro-objects, based on the assayresults obtained in the assay sector. The isolation sector may includeat least one sequestration pen which may be configured like thesequestration pens of the selection sector as described above, but maynot have any capture structures located within the sequestration pen.The isolation sector may include a plurality of sequestration pens. Theisolation sector may be a distinct region in the enclosure of themicrofluidic device that is fluidically connected to the selectionsector. The isolation sector may further include a microfluidic channelwhich is part of the flow region, and wherein each of the at least onesequestration pens opens off of the microfluidic channel. The opening ofthe at least one sequestration pen of the isolation sector may openlaterally from the microfluidic channel.

Polymers for Use in the Solidified Polymer Network of the InSitu-Generated Capture Structure.

In various embodiments of the solidified polymer network of an insitu-generated capture structure, the solidified polymer network may bea synthetic polymer, a modified synthetic polymer, or a light ortemperature activatable biological polymer. The functionalizedpre-polymer used to form the solidified polymer network may be any ofthe polymers described herein for use within the solidified polymernetwork. The biological polymer may be configured to be temperature orlight activatable to form a solidified polymer network. In someembodiments, the biological polymer may be modified to incorporatemoieties providing the ability to be temperature or light activatable.The synthetic polymer modifications may include size modificationmotifs, cleavage motifs, reactive terminal moieties, and/or cellrecognition motifs.

In some embodiments of the solidified polymer network of an insitu-generated capture structure, the solidified polymer network mayinclude at least one of a polyethylene glycol, modified polyethyleneglycol, polylactic acid (PLA), modified polylactic acid, polyglycolicacid (PGA), modified polyglycolic acid, polyacrylamide (PAM), modifiedpolyacrylamide, poly-N-isopropylacrylamide (PNIPAm), modifiedpoly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinylalcohol, polyacrylic acid (PAA), modified polyacrylic acid,polycaprolactone (PCL), modified polycaprolactone, fibronectin, modifiedfibronectin, collagen, modified collagen, gelatin, modified gelatin,laminin, modified laminin, polysaccharide, modified polysaccharide, or aco-polymer in any combination. In other embodiments, the polymer mayinclude at least one of a polyethylene glycol, modified polyethyleneglycol, polylactic acid (PLA), modified polylactic acid, polyglycolicacid (PGA), modified polyglycolic acid, polyvinyl alcohol (PVA),modified polyvinyl alcohol, polyacrylic acid (PAA), modified polyacrylicacid, polycaprolactone (PCL), modified polycaprolactone, fibronectin,modified fibronectin, collagen, modified collagen, laminin, modifiedlaminin, polysaccharide, modified polysaccharide, or a co-polymer in anycombination. In yet other embodiments, the polymer may include at leastone of a polyethylene glycol, modified polyethylene glycol, polylacticacid (PLA), modified polylactic acid, polyglycolic acid (PGA), modifiedpolyglycolic acid, polyvinyl alcohol (PVA), modified polyvinyl alcohol,polyacrylic acid (PAA), modified polyacrylic acid, fibronectin, modifiedfibronectin, collagen, modified collagen, laminin, modified laminin, ora co-polymer in any combination. In some embodiments, the solidifiedpolymer network does not include a silicone polymer. In someembodiments, the solidified polymer network may not include a polylacticacid (PLA) or a modified polylactic acid polymer. In other embodiments,the solidified polymer network may not include a polyglycolic acid (PGA)or a modified polyglycolic polymer. In some embodiments, the solidifiedpolymer network may not include a polyacrylamide or a modifiedpolyacrylamide polymer. In yet other embodiments, the solidified polymernetwork may not include a polyvinyl alcohol (PVA) or a modifiedpolyvinyl alcohol polymer. In some embodiments, the solidified polymernetwork may not include a polyacrylic (PAA) or modified PAA polymer. Insome other embodiments, the solidified polymer network may not include apolycaprolactone (PCL) or a modified polycaprolactone polymer. In otherembodiments, the solidified polymer network may not be formed from afibronectin or a modified fibronectin polymer. In some otherembodiments, the solidified polymer network may not be formed from acollagen or a modified collagen polymer. In some other embodiments, thesolidified polymer network may not be formed from a laminin or amodified laminin polymer. In some embodiments, the solidified polymernetwork may include only one kind of polymer. In various embodiments,the solidified polymer network including only one kind of polymerincludes modified polyethylene glycol polymer.

Physical and chemical characteristics determining suitability of apolymer for use in the solidified polymer network may include molecularweight, hydrophobicity, solubility, rate of diffusion, viscosity (e.g.,of the medium), excitation and/or emission range (e.g., of fluorescentreagents immobilized therein), known background fluorescence,characteristics influencing polymerization, and pore size of asolidified polymer network. The solidified polymer network is formedupon polymerization or thermal gelling of a flowable polymer (e.g., apre-polymer solution,)

One type of polymer, amongst the many polymers that may be used, ispolyethylene glycol diacrylate (PEGDA), which is a member of the groupof modified polyethylene glycol polymers. The mechanism of lightinitiated polymerization is shown in Equation 1. The free radicalinitiator Igracure® 2959 (BASF), a highly efficient, non-yellowingradical, alpha hydroxy ketone photoinitiator, is typically used forinitiation at wavelengths in the UV region (e.g., 365 nm), but otherinitiators may be used. An example of another useful photoinitiatorclass for polymerization reactions is the group of lithium acylphosphinate salts, of which lithium phenyl 2, 4,6,-trimethylbenzolylphosphinate has particular utility due to its moreefficient absorption at longer wavelengths (e.g., 405 nm) than that ofthe alpha hydroxy ketone class.

Equation 1.

Other types of PEG that may be photopolymerized include PEGdimethylacrylate, and/or multiarm PEG (n-PEG) acrylate (n-PEG-Acr).Other polymer classes that may be used include poly vinyl alcohol (PVA),polylactic acid (PLA) polyacrylic acid (PAA), polyacrylamide (PAM),polyglycolic acid (PGA) or polycaprolactone (PCL).

The molecular weight range of the polymer may be varied as required forthe performance of the disclosed in situ-generated capture structures. Awide range of molecular weights of the flowable polymer may be suitable,depending upon the structure of the polymer. A useful star type polymermay have M_(w) (weight average molecular weight) in a range from about500 Da to about 20 kDa (e.g., four arm polymer), or up to about 5 kDafor each arm or for a linear polymer, or any value therebetween.

Various co-polymer classes may be used, including but not limited to:any of the above listed polymer, or biological polymers such asfibronectin, collagen or laminin. Polysaccharides such as dextran ormodified collagens may be used. Biological polymers havingphotoactivatable functionalities for polymerization may also be used.

Crosslinking may be performed by radiation of linear or branched PEGpolymers, free radical polymerization of PRG acrylates, and specificallytailored chemical reactions such as Michael addition, condensation,Click chemistry, native chemical ligation and/or enzymatic reactions.

The polymers may be selected to have a desired range of crosslinkingbased on the nature of the polymer (configuration of the flowablepolymers such as star, multiarm or comb polymers, length of polymersegments between crosslinkable functionalities) and polymerizationconditions (extent of temperature or photoinitiation, amount ofphotoactivatable initiator present, amount of radical terminator speciespresent, and the like).

In some embodiments, the polymer of the solidified polymer network maybe a modified PEG polymer. The polymer may be a star, 4-arm or 2-arm PEGdiacrylate polymer.

Swellable Polymers.

PEG polymers may be swellable under various conditions and may bereversed by reverting back to the original media/temperature.Poly-N-isopropylacrylamide (PNIPAm) may be swelled by increasingtemperature, and de-swelled by cooling.

Size Modification Motifs.

Some hydrogels, including poly-N-isopropylacrylamide (PNIPAm) or polyacrylamide (PAM), may also incorporate specific moieties such asazobenzene which changes cis/trans orientation upon exposure to light atthe surface of the functionalized polymer. This shift can providesignificant change in size of the portion of polymer such as an insitu-generated capture structure within a pen. These polymers mayalternatively include cinnamic acid functionalities that cross link uponexposure to UV light, which is reversible upon removal of the light. Thecross-linked polymer is elongated compared to the non-crosslinked state.Another moiety which may be introduced to these polymers includestriphenyl leucomethane, which forms ion pairs upon application of light,reversibly, upon exposure to light. The wavelength of activating lightcan be brought into the visible range if trisodium copper chlorophyllinis incorporated into the polymer.

Other Modifications for Functionalization.

A polymer (e.g., PEG) may be modified by incorporating functional groupsat one or both of the termini of the (PEG) polymer, which may includethiol, maleimide, carboxyl, amine, methoxy, azide, vinyl sulfone,acetylenic, or acrylate functionalities. The functional groupsintroduced may be the same or different. Desired peptide motifs,antibodies, or other defined molecular functionalization by appropriatechemical elaboration may be introduced so that the moiety is capably ofreacting specifically with a corresponding functionality on the polymer.Biotinylation may be introduced for later reaction with another specieslinked to streptavidin, or vice versa.

The polymer may include various motifs, including cleavage motifs,reactive terminal motifs, or cell recognition motifs. A cleavage motifmay include a peptide sequence inserted into the polymer that is asubstrate for one or more proteases, including but not limited to amatrix metalloproteinase, a collagenase, or a cysteine protease such asa caspase or cathepsin. Another category of cleavage motif may include aphotocleavable motif such as a nitrobenzyl photocleavable linker whichmay be inserted into selected locations of the prepolymer. A cleavagemotif may be utilized either to remove the solidified polymer network ofan in situ-generated capture structure or may be used, as described, asthe assay analyte itself when incorporated within an in situ-generatedcapture structure. The polymer may be modified to incorporate chemicallyreactive motifs such as, but not limited to, N-hydroxysuccinimidyl(NHS), biotin, alkynyl, or azido moieties, which may be used tointroduce assay reagents or assay analytes to the solidified polymernetwork of the in situ-generated capture structure as described above.In other embodiments, the polymer may include cell recognition motifsincluding but not limited to a RGD peptide motif, which is recognized byintegrins, or a furin substrate as described above, which may be used asassay analyte or assay reagents.

Reversing/Removing/Minimizing the In Situ-Generated Capture Structure.

A number of mechanisms may be used to remove or reduce the insitu-generated capture structure when there is no further purpose forit. For example, once an assay is completed and desirable biologicalcells have been identified, it may be useful to remove the insitu-generated capture structure in order to continue culturing andexpanding the biological cell demonstrating desirable activities orproperties.

Mechanical force. Increasing flow can be used if at least a portion ofthe in situ-generated capture structure is located within a flow regionas opposed to an isolation region of a pen. For example, when the insitu-generated capture structure is located within a flow region of theenclosure, a rate of fluidic flow may be increased through the flowregion which may detach the at least one capture structure from surfacesto which it is attached, including but not limited to the substrate, thewalls of the flow region (which may be formed from microfluidic circuitmaterial), or a cover. In some embodiments, the at least one insitu-generated capture structure may be located within an isolationregion of a sequestration pen, and after the assay is complete, thesequestration pen or the isolation region therein may be modified tobring flow through the isolation region, similarly detaching the insitu-generated capture structure from surfaces it is attached.

Hydrolytic susceptibility: Porogens, which may include polymersincapable of being chemically linked to the photoinitiated polymer(s),may be included when forming the in situ-generated capture structure.The degree/size of openings within the formed hydrogel can customize thehydrolysis rate via accessibility within the in situ-generated capturestructure). In other embodiments, the pores formed may be employed topermit secreted materials or chemical reagents to pass through the insitu-generated capture structure but prevent a cell from moving throughthe in situ-generated capture structure. In other embodiments,degradability of these polymers may be increased by introducingdegradable segments such as polyester, acetal, fumarate, poly(propylenefumarate) or polyhydroxyacids into polymers (e.g., PEG polymers).

Reducing agents: PEG may be formed with disulfide linkages at intervalsalong the macromere, which may be random or predetermined. The disulfidebonds may be broken by Dithiothreitol (DTT), mercaptoethanol, or TCEP.

Thermal: poly N-isopropylacrylamide (PNIPAm) or other suitable LCSTpolymers may be used to introduce capture structures upon heating. Theymay be removed by decreasing the temperature of the formed polymercapture structure. The polymers may include ELPs or other motifs thatalso permit removal by other mechanisms such as hydrolysis orproteolysis. In particular, PNIPAm may be used to create a surface foradherent cells, but then switched to permit export. Other polymers mayalso demonstrate size change depending on temperature. For example,PEGDA hydrogel incorporated within an in situ-generated capturestructure may swell by about 20% upon cooling from a temperature ofabout 40° C. to about 20° C. The size of a PEGDA hydrogel insitu-generated capture structure may be increased locally by using laserillumination directed at the substrate underlying the in situ-generatedcapture structure or at a transparent cover over the in situ-generatedcapture structure, which may be, for instance ITO.

Proteolytic susceptibility: Hydrogels may have any sort of peptidesequence engineered in, such that selective proteolysis upon a selectedmotif by a selected protease can remove/reverse/or minimize a hydrogelcapture structure. Some classes of modified PEG include PEG havingelastin like peptide (ELP) motifs and/or having peptide motifs forsusceptibility to a variety of proteases (enzyme sensitive peptide ESP).A large number of these motifs are known. One useful motif is RGD whichmay be constrained to be cyclic.

Osmotic susceptibility: Calcium concentration/other osmotic strategiescan be employed to degrade and remove an in situ-generated capturestructure. As above, dimensionally swell or de-swell capture structuresusing media changes.

Light initiated photocleavage: As described above, a photocleavablemotif such as a nitrobenzyl photocleavable linker may be incorporatedwithin the solidified polymer network of an in situ-generated capturestructure. An in situ-generated capture structure including aphotocleavable motif may be susceptible to removal or reduction in sizeby exposure to light including at least some light having a suitablewavelength to initiate cleavage of the photocleavable motif.

In some applications, the in situ-generated capture structure may not beremoved but may simply be swelled or de-swelled using light ormedia\solvent changes. Some types of hydrogels may incorporate moietiesthat respond reversibly to light (for example, change regiochemistryabout a rigid bond; form reversible crosslinks within the polymer, orform/break ion pairs).

Microfluidic Device Assisted Heating.

The microfluidic device may further include a metal pad disposed on thesubstrate at a location of the in situ-generated capture structure. Themetal pad may be created by deposing a contiguous metal shape or apattern of metal shapes onto the substrate. The thermal pad can compriseany type of metal that can be excited by a light source to produce heat.Suitable metals include chromium, gold, silver, aluminum, indium tinoxide, or any combination thereof. Metals may be combined in amulti-layered thermal pad, e.g., a layer of chromium, a layer oftitanium, a layer of gold. Other metals (and alloys) are known in theart. The thermal pad can comprise a continuous metal surface or cancomprise a pattern of metal (e.g. metal shapes such as dots, squares,lines, cones, irregular forms). In some embodiments, a gold pad may bedisposed on the substrate at a location where an in situ-generatedcapture structure will be/has been generated. The thermal pad may beused to generate heat to gel, swell, reduce, or remove an insitu-generated capture structure. Heat may be generated by directinglight into the microfluidic device at the location where such gelling,swelling, reduction or removal is desired. In some embodiments, thesolidified polymer network may include a thermosensitive polymer. When asolidified polymer network of an in situ-generated capture structureincludes a thermosensitive polymer, the device may further include athermal pad disposed on the substrate at a location beneath the at leastone in situ-generated capture structure will be introduced.

Methods of Assaying a Micro-Object Using Functionalized CaptureStructures.

A method is provided for assaying a micro-object (e.g., a biologicalcell or an embryo) or a biological product produced by the micro-object,in a microfluidic device having at least a first in situ-generatedcapture structure including steps of: disposing a micro-object withinthe microfluidic device in a region proximal to the first insitu-generated capture structure, where the in situ-generated capturestructure includes a solidified polymer network, and further where thesolidified polymer network includes an assay reagent or an assayanalyte; contacting the assay reagent or assay analyte with themicro-object or a biological product of the micro-object; and detectingan interaction of the assay reagent or assay analyte with themicro-object or the biological product. In various embodiments, a methodis provided for assaying a micro-object (e.g., a biological cell) in amicrofluidic device having at least a first in situ-generated capturestructure including steps of: disposing a micro-object within amicrofluidic device in a region proximal to the t in situ-generatedcapture structure, where the in situ-generated capture structureincludes a solidified polymer network, and further where the solidifiedpolymer network includes an assay analyte; contacting the assay analytewith a biological product of the micro-object; and detecting aninteraction of the assay analyte with the biological product.

In yet other embodiments, another method is provided for assaying amicro-object (e.g., a biological cell) in a microfluidic device havingat least a first in situ-generated capture structure including steps of:disposing a micro-object within a microfluidic device in a regionproximal to the first in situ-generated capture structure, where the insitu-generated capture structure includes a solidified polymer network,and further where the solidified polymer network includes an assayanalyte; contacting the assay analyte with the micro-object; anddetecting an interaction of the assay analyte with the micro-object. Inyet other embodiments, a method is provided for assaying a micro-object(e.g., a biological cell) in a microfluidic device having at least afirst in situ-generated capture structure including steps of: disposinga micro-object within a microfluidic device in a region proximal to thefirst in situ-generated capture structure, where the in situ-generatedcapture structure includes a solidified polymer network, and furtherwhere the solidified polymer network includes an assay reagent;contacting the assay reagent with the micro-object or a biologicalproduct of the micro-object; and detecting an interaction of the assayreagent with the micro-object or the biological product.

The assay reagent or assay analyte of any of the above methods mayinclude any assay reagent or assay analyte described herein.

In various embodiments of any of the methods of assaying a micro-object,the assay reagent or assay analyte that is included within and/or on thein situ-generated capture structure may be covalently or non-covalentlyattached to the solidified polymer network of the in situ-generatedcapture structure, in any way described above. The assay reagent orassay analyte may include a protein, an oligonucleotide, an organicmolecule, or a saccharide. The assay reagent or assay analyte may be,respectively, any assay reagent or assay analyte as described above.

In various embodiments of any of the methods of assaying a micro-object,the biological product of a micro-object may include a protein, anoligonucleotide, an organic molecule, or a saccharide. The biologicalproduct of the micro-object may be any biological product as describedherein, and may function in the methods as the analyte measured by anassay reagent included within and/or on the in situ-generated capturestructure. Alternatively, the biological product of the micro-object maybind to, react with, and/or cleave an assay analyte included withinand/or on the in situ-generated capture object, whereby the biologicalproduct acts as a reagent in the assay method.

In various embodiments of any of the methods of assaying a micro-object(e.g., a biological cell or embryo), the microfluidic device may includean enclosure comprising: a substrate, microfluidic circuit materials anda flow region located within the enclosure, where the at least one insitu-generated capture structure is disposed within the enclosure. Insome embodiments, the enclosure of the microfluidic device furtherincludes at least one sequestration pen, where the at least one insitu-generated capture structure may be disposed within the at least onesequestration pen. The sequestration pen may include an isolation regionand a connection region, where the connection region may have a proximalopening to the flow region and a distal opening to the isolation region.In some embodiments, the in situ-generated capture structure may bedisposed within the isolation region of the sequestration pen. The flowregion may include a channel. The at least one capture structure may bedisposed at a first location adjacent to a first wall of thesequestration pen.

In various embodiments of any of the methods of assaying a micro-object,the enclosure may include a plurality of sequestration pens. Theplurality of sequestration pens may be aligned in a row, and theproximal opening of each of the plurality of sequestration pens may openin a common direction within the flow region. In some embodiments, theflow region may include a channel and the proximal opening of each ofthe plurality of sequestration pens may open off one side of themicrofluidic channel.

In various embodiments of any of the methods of assaying a micro-object,the substrate of the microfluidic device may be configured to generate adielectrophoretic (DEP) force upon a micro-object in a fluidic mediumwithin the enclosure. The step of disposing the micro-object(s), (e.g.,biological cell(s)), within the microfluidic device to the regionproximal to the at least first in situ-generated capture structure mayinclude moving the micro-object(s) using dielectrophoretic force. Thedielectrophoretic force may be optically actuated. Alternatively, thesubstrate of the microfluidic device may be configured to generate anelectro-wetting force on a droplet within the enclosure. The step ofdisposing the micro-object(s) within the microfluidic device to theregion proximal to the at least first in situ-generated capturestructure may include moving the micro-object(s) using electrowettingforce. The electrowetting force may be optically actuated. In someembodiments, the microfluidic device may include one or more substrateswhich may be configured to generate both dielectrophoretic forces andelectrowetting forces within the enclosure of the microfluidic device,each of which may be light actuated. Alternatively, fluidic flow withinthe flow region (e.g., microfluidic channel) and/or gravity may be usedto dispose the micro-object(s) within the microfluidic device. In someembodiments, a combination of dielectrophoretic forces, electrowettingforces, gravity and/or fluidic flow may be used to dispose themicro-object(s). In various embodiments, the microfluidic device mayhave at least one surface that includes a coating material. In someembodiments, the coating material may covalently modify the at least onesurface to provide a conditioned surface which enhances cell growth,viability, portability and any combination thereof. The conditionedsurface may be selected to be any suitable conditioned surface describedherein.

In various embodiments of any of the methods of assaying a micro-object,the assay reagent or assay analyte included within and/or on the insitu-generated capture structure is allowed to interact with themicro-object (e.g., biological cell or embryo) or biological product ofthe micro-object. In some embodiments, the interaction may benon-covalent, e.g., binding of a protein such as an antigen or acytokine (e.g., an analyte), which is a biological product of themicro-object, with an antibody (e.g., an assay reagent) included withinand/or on the in situ-generated capture structure. In another example ofa noncovalent interaction, the in situ-generated capture structure mayinclude a binding recognition motif for a protein expressed on thesurface of a micro-object, where if the micro-object expresses theprotein of interest, it may be bound to the in situ-generated capturestructure. In other embodiments, chemical bonds may be cleaved by theinteraction, for example when the in situ-generated capture structureincludes a protease recognition motif, thereby providing an assayanalyte bound to the in situ-generated capture structure. In thisembodiment, the interaction may be the interaction of a protease eithersecreted by a micro-object or expressed on the surface of themicro-object, which interacts with the recognition motif to cleave thesubstrate incorporated within the in situ-generated capture structure.In yet other embodiments, the interaction may include a covalentinteraction. For example, the in situ-generated capture structure mayinclude an assay reagent such as an antibody configured to bindspecifically with a biological product (e.g., antigen, cytokine, or anysecreted biological product), where the in situ-generated capturestructure or the antibody also includes a reactive moiety such as acrosslinking moiety (e.g., carboxylic acid, amino moiety, thiol moiety,or activated species thereof) which covalently binds the biologicalproduct that non-covalently binds to the antibody assay reagent.

In various embodiments of any of the methods of assaying a micro-object,the step of detecting comprises detecting a signal from the at least onecapture structure. In various embodiments of the method, the detectablesignal may be incorporated within an assay reagent or assay analyteincluded within and/or on the in situ-generated capture structure. Thedetectable signal may be concentrated to a surface of the at least onecapture structure or may be detected in a region immediately adjacent tothe in situ-generated capture structure within the enclosure oralternatively, within the sequestration pen. In yet other embodiments,the detectable signal may be concentrated to an interior portion of thein situ-generated capture structure, which may occur via diffusionthroughout the solidified polymer network. The detectable signal may befluorescent, luminescent or colorimetric. In some embodiments, thesignal may be fluorescent. In some embodiments, the step of detectingthe fluorescent signal may further include quantifying the fluorescentsignal.

In some embodiments of any of the methods of assaying a micro-object,the step of detecting a signal from the at least one capture structuremay include detecting loss of an initial fluorescent signal of the atleast one capture structure. For example, an in situ-generated capturestructure may include a solidified polymer network that comprises aprotease substrate motif as an assay analyte, where the substrate motifincludes a detectable signal such as a fluorescent label. Detecting theinteraction between the assay analyte and a protease capable of cleavingit may include detecting the extent of loss of fluorescent signal fromthe in situ-generated capture structure incorporating the fluorescentprotease substrate motif. In another embodiment, the method may includedetecting a gain of fluorescent signal, when the in situ-generatedcapture structure incorporating a protease substrate motif as an assayanalyte includes a quenched fluorescent pair (e.g., a FRET pair, whichmay have dual labels on suitably spaced different amino acids in theinserted substrate motif, or may include a molecular beacon or otherFRET probe construct). In this embodiment, the interaction between theprotease substrate motif of the in situ-generated capture structure anda protease produced by a micro-object (e.g., the biological product) maypermit detection of an increase of signal when cleavage of the substrateby the protease increases the spatial separation between the quenchedfluorescent pair. One or more fluorescent signals may be detected.

In other embodiments of any of the methods of assaying a micro-object,the fluorescent signal may be incorporated within the biological productor micro-object that interacts with the assay reagent or assay analyteincluded within and/or on the in situ-generated capture structure. Forexample, a protein of interest secreted by a micro-object (e.g.,biological product) may also include a signal such as green fluorescentprotein (GFP), and thus may be directly detectable when interacting withan antibody included within and/or on the in situ-generated capturestructure that binds specifically to the protein of interest. In otherembodiments, a micro-object expressing a protein of interest on itssurface may also have a detectable signal that is contained within themicro-object (such as mCherry, an inserted protein having a sequencerelated to Discoma sp.), whereupon binding with the antibody includedwithin and/or on the in situ-generated capture structure (e.g., assayreagent), fluorescence of the intracellular protein may be directlydetected.

In yet other embodiments of any of the methods of assaying amicro-object, the step of detecting further includes introducing adetection reagent having a detectable label to the region proximal tothe in situ-generated capture structure. Any suitable detection reagentdescribed herein may be used. Introduction of the detection reagent mayinclude flowing a solution containing the detection reagent through theflow region of the microfluidic device. In some embodiments, when the atleast one in situ-generated capture structure is located within asequestration pen, the detection reagent may enter the sequestration pencontaining the in situ-generated capture structure substantially or onlyby diffusion, after being flowed into the enclosure of the microfluidicdevice. The introduction of the detection reagent may be performed afterthe step of disposing the micro-object into the enclosure oralternatively, into the sequestration pen, has been performed. In someembodiments, the detection reagent may be introduced before themicro-object has been introduced to the enclosure or the sequestrationpen. In some embodiments, the step of introducing the detection reagentmay be performed just prior to performing the step of detecting. Invarious embodiments, the detection reagent is configured to beconcentrated to the in situ-generated capture structure when an assayreagent included within and/or on the in situ-generated capturestructure interacts with the biological product or the micro-objectitself (e.g., the analyte of the assay). The detection reagent may beconcentrated to the in situ-generated capture structure (e.g.,constrained to the immediate region of the in situ-generated capturestructure) by any suitable mechanism such as forming a binding pair,hybridizing with a target oligonucleotide, intercalating or covalentlyreacting with the biological product or micro-object, which is itselfconstrained to the immediate region of the in situ generated capturestructure by virtue of the interaction between the assay reagent orassay analyte/micro-object or biological product thereof. In someembodiments, the interaction between the detection reagent/micro-objector biological product thereof/assay reagent or assay analyte/solidifiedpolymer network of the in situ-generated capture structure may form acomplex, which may act to concentrate the detectable signal of thedetection reagent. In some embodiments, the detectable label of thedetection reagent may not be detectable until it is concentrated to thein situ-generated capture structure. For example, if the detectionreagent is an intercalator dye or molecular beacon (Fret quenchedhairpin probe), Scorpions (Fret quenched probe/primer combination) orany other FRET label of an oligonucleotide, detecting the signal may notbe performed until after the detection reagent binds to the biologicalproduct of the micro-object or the micro-object itself, which in someembodiments, may be immobilized to the in situ-generated capturestructure. In various embodiments, the detectable label of the detectionreagent is non-covalently attached to the assay reagent or assayanalyte.

In some embodiments of any of the methods of assaying a micro-object,the detection reagent comprises at least a first antibody. One or moreantibodies may be used to detect the interaction between the assayreagent or assay analyte included within and/or on the in situ-generatedcapture structure and the biological product of the micro-object or themicro-object itself. In some embodiments, the method may includeintroducing a first detection antibody having specificity for the assayanalyte/(biological product or micro-object) pair or the assayreagent/(biological product or micro-object) pair, followed byintroducing a second antibody that is labeled and can bind to at leastone portion of the assay analyte/(biological product or micro-object)pair or the assay reagent/(biological product or micro-object) pair. Insome embodiments, the method may include introducing a second antibodyhaving specificity for the first detection antibody. In otherembodiments, the labeled second antibody may have specificity for acomplex of the assay analyte/(biological product or micro-object) pairor a complex of the assay reagent/(biological product or micro-object)pair.

In various embodiments of any of the methods of assaying a micro-object,the method may further include a step of exporting the micro-object fromthe microfluidic device. The micro-object may be exported based on theresults of the assay (e.g., demonstrating a desirable level of signal inthe detection step). In various embodiments of the method, exporting themicro-object from the microfluidic device may further include moving themicro-object to another portion of the substrate of the microfluidicdevice. For example, the steps of assaying may be performed within aselection sector as described herein, and micro-objects demonstratingselected characteristics as identified by the method of assaying, may bemoved by DEP forces, electro-wetting forces, gravity or fluidic flow toan isolation sector of the microfluidic device for further processing.

In various embodiments of any of the methods of assaying a micro-object,the method may further include a step of reducing or removing the insitu-generated capture structure by introducing a hydrolytic agent,introducing a proteolytic agent, introducing a fluidic medium thatincreases or decreases osmolality of the fluidic medium within the flowregion and/or the sequestration pen, changing temperature of the insitu-generated capture structure, or optically illuminating the insitu-generated capture structure, thereby reducing or removing the atleast one capture structure. The step of changing the temperature mayfurther include optically illuminating a thermal pad on the substrateadjacent to or under the in situ-generated capture structure. In someembodiments, reducing (e.g., reducing the size or number offunctionalized sites of the in-situ generated capture structure) orremoving the in situ-generated capture structure may release amicro-object from its concentration/constraint to the in situ-generatedcapture structure.

In other embodiments, the step of exporting a micro-object may includeintroducing a competing binding partner for the assay reagent/assayanalyte to which the micro-object has bound, as described above. Thecompeting binding partner for the assay reagent/assay analyte may causethe micro-object to be released from its binding interaction with thearray reagent/assay analyte and permit export of the micro-object.

FIGS. 5A-E shows one embodiment of the method of assaying a micro-object(e.g., a biological cell or an embryo) in a microfluidic device havingat least a first in situ-generated capture structure within anenclosure. In this embodiment, the enclosure includes a sequestrationpen including the at least a first capture structure disposed therein,where the in situ-generated capture structure acts as a pre-selectedassay region. The in situ-generated capture structures may befunctionalized with, for example an assay reagent, as shown in FIG. 5A.The method is not so limited, and the in situ-generated capturestructure may be functionalized to contain an assay analyte instead.

In FIG. 5A, an in situ-generated capture structure 502 is shown, whichhas, for example, streptavidin introduced into the solidified polymernetwork. Prepolymer solutions of the structural polymer (e.g., astreptavidin modified reactive prepolymer) and soluble initiator may beflowed into the microfluidic device. Precise and selectivesolidification of the in situ-generated capture structure can beaccomplished by illumination in one corner of the sequestration pen 530,similarly to the schematized process shown in FIG. 4C for thetransformation of prepolymer 405 to in situ-generated capture structure404. In this embodiment, the in situ-generated capture structure 502 isgenerated to be located near/at a wall formed of microfluidic circuitmaterial 260 at a corner of the isolation region distal to the openingof the sequestration pen 530 into the microfluidic channel 264, wherefluidic medium flows (278). After formation of the in situ-generatedcapture structure, excess polymer solution and initiator may be removedfrom the system by flushing the microfluidic channel 264, and permittingthe unused reagents to diffuse out of the sequestration pen 530. The insitu-generated capture structure 502 may present streptavidin both onthe surface of the in situ-generated capture structure, and throughoutthe solidified polymer network.

A functionalized antibody may be introduced to the isolation region ofthe sequestration pen 530. The functionalized antibody may have a biotinfunctionality, which may bind to the streptavidin sites on the surfaceof or within the in situ-generated capture structure 502, therebyproviding antibody 504 included at the surface or within the insitu-generated capture structure 502, providing an in situ-generatedcapture structure (502 plus 504) equivalent to capture structure 406 ofFIG. 4B (which is also equivalent to schematic in situ-generated capturestructure 406B of FIG. 4C). The antibody 504 may be any antibody that isspecific for a secreted biological product. In some embodiments, theantibody 504 may be a cytokine, such as IL-2, IFN alpha/beta, TNF alpha,and the like. The antibody 504 may be used to detect cells that secretethe cytokine of interest. All of the biotinylated antibody 504 does notneed to be included within and/or on the at least one in situ-generatedcapture structure. Some portion of the biotinylated antibody 504 mayalso be free-floating in the solution. The reverse pairing may also beused, e.g., the in situ-generated capture structure 502 may havebiotinylated sites incorporated by the photoinitiated solidification ofthe polymer network, and the antibody 504 may be modified to includestreptavidin. The streptavidin functionality can bind to the biotinsites on the in situ-generated capture structure 502, thereby alsoproviding antibody 504 included at the surface or within the insitu-generated capture structure 502.

While in FIGS. 5A-5E, the assay reagent is shown as an antibody forconvenience and simplicity, the method is not so limited. The assayreagent may be any suitable assay reagent as described herein.

Biological cells 506 can be flowed into the microfluidic channel 264,and disposed by any suitable method described herein into isolationregions of the sequestration pen 530. Cell(s) 506 of interest may beintroduced into the pen having a streptavidin functionalized capturestructure, before or after the streptavidin functionalized antibody isintroduced. There may be one or more cells 506 of interest. In someembodiments, there may be a single cell 506.

The cell(s) can be cultured, and may secrete a biological product. Thebiological product may be a protein. One non-limiting example of aproteinaceous biological product of a cell may be a cytokine. Onenon-limiting example of a cell that may produce a cytokine may be aT-cell.

As shown in FIG. 5B, as cell culturing continues, the cell 506 mayproduce the biological product 508 that can bind to the antibody assayreagent 504. The biological product 508 will be captured by itsinteraction with the assay reagent 504 on the in situ-generated capturestructure 502, e.g., captured by antibodies incorporated within or onthe surface of the in-situ generated capture structure. FIG. 5C shows anexpanded view of the in situ-generated capture structure 502 of FIG. 5B,showing the complex 510 that is formed by interaction (e.g., binding) ofthe biological product 508 with the assay reagent 504 (e.g., antibody)of the in situ-generated capture structure 502. The in situ-generatedcapture structure 502 can be located either near the proximal opening tothe microfluidic channel within the pen or can be located within a moredistal section of the connection region or within the isolation regionof the pen.

The antibodies, assay reagent 504, included at the surface or within thein situ-generated capture structure 502, capture and concentrate thebiological product of interest 508 (e.g., analyte). The concentratedcaptured biological product 508/antibody 504 of complex 510 may be madedetectable by introduction of the labeled antibody 512, which may befluorescently labeled, as shown in FIG. 5D. Detection of the fluorescentsignal of the immobilized antibody/cytokine/antibody complex 514, whichis concentrated to the solidified polymer network of the insitu-generated capture structure 502/504 can permit detection andranking of more/less actively secreting biological cells. FIG. 5E showsan expanded view of the region of the sequestration pen where the insitu-generated capture structure 502 is located. The complex 514 of theimmobilized antibody 504/cytokine (e.g., biological product 508)/labeledantibody 512 is shown. While the detection reagent is shown here as anantibody for simplicity, the detection reagent is not so limited but maybe any suitable detection reagent as described herein.

In some other embodiments, the biological product 508 may itself containa detectable label, such as, but not limited to green fluorescentprotein. When the biological product 508 includes a detectable label,additional labeling by a detection reagent may not be performed, but theamount of detectable label of the biological product 508 may be directlydetected. Thus, in some embodiments of the method, an analyte (e.g.,biological product 508) may be a detectable analyte and may interact andbe captured by the assay reagent 504 to form a detectable complex 510′(not shown), which may be detected.

Multiplexed Assay Methods.

In various embodiments of methods of assaying one or more micro-objects(e.g., biological cell or embryo), a multiplexed assay may be performed.In one embodiment, the at least one capture structure located within theenclosure, or, optionally, within a sequestration pen, may contain morethan one assay reagent or assay analyte. In other embodiments, theenclosure, or optionally, a sequestration pen therein, may include afirst capture and a second capture structure. The first capturestructure may be as described above and include a first assay reagent orfirst assay analyte. The second capture structure may include a secondsolidified polymer network, and the second solidified polymer networkmay include a second assay reagent or a second assay analyte. The secondsolidified polymer network and the second assay reagent or second assayanalyte may include any feature as described above in any combination.In some embodiments, each of the first and second capture structuresincludes a different assay reagent or assay analyte. In someembodiments, the first and the second capture structures may include afirst assay reagent and a second assay reagent that differ from eachother. In other embodiments, the first and the second capture structuremay contain a first assay analyte and a second assay analyte that differfrom each other. In yet other embodiments, the first capture structureand the second capture structure may include an assay reagent on onecapture structure and an assay analyte on the second capture structure.In some embodiments of the multiplexed assay, the first capturestructure and the second capture structures may be disposed within theenclosure or, alternatively, the sequestration pen, at distinguishablelocations. The distinguishable locations of the first and the secondcapture structure may be adjacent to the same wall of the enclosure orto the same wall of a sequestration pen therein, or adjacent todifferent walls of the enclosure or to different walls of asequestration pen therein.

In various embodiments of the multiplexed assay, the step of detectingincludes detecting a first analyte and a second analyte, wherein thefirst analyte is different from the second analyte. The first analyteand the second analyte may be a first biological product and a secondbiological product secreted by a micro-object. In other embodiments, thefirst analyte and the second analyte may be a biological productsecreted from a micro-object and the second analyte may be a biologicalproduct present on the surface of the micro-object, and may be differentfrom each other. In other embodiments, the step of detecting may includedetecting a first biological product that interacts with a first assayanalyte on the first capture structure and detecting a second biologicalproduct that interacts with a second assay analyte on the second capturestructure. In yet other embodiments, the step of detecting may includedetecting a first biological product that interacts with a first assayreagent and detecting a second biological product that interacts with asecond assay reagent.

The step of detecting the interactions may further include introducing afirst detection reagent and a second detection reagent to the regionproximal to the first and second capture structures, wherein each of thefirst and second detection reagents includes a detectable label. Thedetectable labels of the first detection reagent and the seconddetection reagent may each independently be fluorescent, colorimetric,or luminescent. The step of detecting may further include detecting afirst fluorescent signal of the first detectable label and a secondfluorescent signal of the second detectable label. In some embodiments,the first fluorescent signal and the second fluorescent signal may bephysically distinguishable, e.g., located at different positions withinthe enclosure, or alternatively at different positions within thesequestration pen therein. In other embodiments, the first fluorescentsignal and the second fluorescent signal may be spectrallydistinguishable. In other embodiments, one of the first or the seconddetectable signals may be fluorescent and the other of the first or thesecond detectable signals may be not fluorescent.

In various embodiments of the multiplexed method, each of the first andsecond detectable reagents may be non-covalently attached to therespective first or second assay reagent or assay analyte. In someembodiments, each of the first and second detection reagents may includean antibody. In some embodiments, each of the first and second detectionreagents include a respective third and fourth antibody, each of whichmay have a detectable label, where the third antibody binds specificallyto the first assay reagent or first assay analyte/(biological product ormicro-object) pair and the fourth antibody binds specifically to thesecond assay reagent or assay analyte/(biological product ormicro-object pair. In some embodiments, the third antibody may be asecondary antibody to the first assay reagent when the first assayreagent is an antibody. In some embodiments, the fourth antibody may bea secondary antibody to the second assay regent, when the second assayreagent is an antibody.

In various embodiments of the multiplexed assay, the first and/or thesecond fluorescent signals may be quantified.

In various embodiments of the method, a multiplex assay may be performedon three or more characteristics of the biological product or three ormore different biological products of a micro-object or the micro-objectitself, or any combination thereof. There may be a third or more capturestructure in the enclosure or alternatively within the at least onesequestration pen therein. Each of the third or more capture structuremay include a solidified polymer network, and the solidified polymernetwork of each of the third or more solidified polymer network mayinclude an assay reagent or an assay analyte. The assay reagent or assayanalyte of each of the third or more capture structure may be differentfrom the first assay reagent or assay analyte of the first capturestructure, and/or may be different from the second assay reagent orassay analyte of the second capture structure. Each of the assay reagentor assay analyte of the third or more capture structures may bedifferent from each other. In other embodiments, one or both of thefirst and second capture structures may alternatively include more thanone assay reagent or assay analyte, distinguishable from the first assayreagent or assay analyte of the first capture structure anddistinguishable from the second assay regent or assay analyte of thesecond capture structure.

In various embodiments of a multiplexed method of assaying one or moremicro-objects, the step of detecting further comprises detecting afirst, a second, a third or more detectable signals that are distinct inlocation within the at least sequestration pen, detectably spectrallydistinct, or a combination thereof.

The multiplexed method may include any of the steps described above forthe singleplex method, including but not limited to disposing the one ormore micro-objects within the microfluidic device where the microfluidicdevice includes at least one capture structure configured to assay formore than one characteristic within the enclosure or within asequestration pen therein, or alternatively includes more than onecapture structure within the enclosure or more than one capturestructure within at least one sequestration pen, where each of the morethan one capture structure is configured to assay for onecharacteristic; allowing a micro-object to release or produce one ormore biological products (any combination of which may be used in anycombination with an assay reagent or assay analyte of any of themultiplexed assay reagents and/or assay analytes as described above);allowing assay analyte(s) or assay reagent(s) to interact with thebiological product(s) or the micro-object itself; and any aspect ofdetecting the interaction.

FIG. 7 illustrates a multiplexed assay according to the methodsdescribed herein, and displays a flow region (microfluidic channel 264)within the enclosure (not shown), and microfluidic circuit material 260,which forms walls defining the channel 264. One sequestration pen 730within microfluidic device 700 is shown, and has walls enclosing thesequestration pen made of microfluidic circuit material 260. asdescribed above. Sequestration pen 730 has three capture structures 702,704, 708 disposed in three physically distinguishable locations withinthe pen 730. There are two micro-objects 706 loaded into the pen 730,which, in this embodiment, are producing biological products 716, 718,and 720. The biological products 716, 718, 720 may be all different ormay be the same biological product which is assayed for three differentcharacteristics by the assay reagents 710, 712, 714 which are includedrespectively within and/or on capture structures 702, 704, 708. Theassay reagents 710, 712, 714 are each different from each other and testfor either a different biological product or a different characteristicof a biological product. As shown in FIG. 7, the assay reagents 710,712, 714 are shown as antibodies for ease of viewing, but the method isnot limited to antibody assay reagents, but may be any suitablecombination of assay reagents and/or assay analytes as described herein.The timepoint illustrated in FIG. 7 is the point in time after themicro-objects 706 have been allowed to produce biological product(s)716, 718, 720; biological product(s) 716, 718, 720, have alreadyinteracted with the assay reagents 710, 712, 714, each of which isimmobilized to respective capture structures 702, 704, 708; and afterthe time point where the detection reagents 722, 724, 726 have alreadybeen introduced to the sequestration pen 730 and have bound specificallyto their targets 716, 718, 720. For ease of viewing, detection reagents722, 724, 726 are represented as antibodies, but the method is notlimited to antibody detection reagents as discussed above. Also, forease of viewing detection reagents 722, 724, 726 each include a label(not shown), where the label may be a detectable label directly attachedto detection reagents 722, 724, 726, or alternatively, the label may beattached to a second antibody (not shown), which binds specifically tothe antibodies 722, 724, 726. Each detectable label directly attached todetection reagents 722, 724, 726 may be detectably distinguishable fromeach of the other detectable labels by being spectrally distinguishableor may be detectably distinguishable by location of the insitu-generated capture structure to which the detectable label of thedetection reagent binds. Each of the assay complexes, which may includea combination of capture structure, assay reagent or assay analyte(e.g., depending on the assay being performed), micro-object orbiological product (e.g., whether the assay reagent or assay analyteinteracts with the micro-object or a biological product of themicro-object), and detection reagent, and shown in FIG. 7 as(702/710/716/722); (704/712/718/724); and/or (708/714/720/726), may bedetected independently or may be detected at the same time. Thedetectable signals from each complex may be quantified, by comparison toan in situ standardized signal, by normalization to each other, and/orany suitable method of quantification.

Methods of Loading.

Loading of biological micro-objects (e.g., biological cells) ormicro-objects (including but not limited to beads) into the enclosure oralternatively to the sequestration pen, can involve the use of fluidflow, gravity, a dielectrophoresis (DEP) force, electrowetting, amagnetic force, or any combination thereof as described herein. The DEPforce can be generated optically, such as by an optoelectronic tweezers(OET) configuration and/or electrically, such as by activation ofelectrodes/electrode regions in a temporal/spatial pattern. Similarly,electrowetting force may be provided optically, such as by anopto-electro wetting (OEW) configuration and/or electrically, such as byactivation of electrodes/electrode regions in a temporal spatialpattern.

Method of Preparation.

A method is provided for preparing at least one capture structure withina microfluidic device. In situ-generated capture structures, may beintroduced either before or after introduction of cells to themicrofluidic (or nanofluidic) device. The in situ-generated capturestructures may be designed to be temporary or may be kept in place untilthe conclusion of the experiment/assay/sorting/culturing process.

The in situ-generated capture structures may be introduced byphotoactivation, temperature change, or osmotic change which can cause apolymer solution present within the microfluidic to form an insitu-generated capture structure capable of preventing a biological cellor a bead from crossing the in situ-generated capture structure.Depending on the mesh size of the in situ-generated capture structure,different categories of chemical species may be permitted to passthrough the in situ-generated capture structure. If the mesh size ischosen to be about 2 nm, only small molecule components may be permittedto pass, but proteins, etc. may sequestered by the in situ-generatedcapture structure. The in situ-generated capture structure may include acrosslinked polymer having a larger mesh size that may not preventsmaller substances such as proteins, nucleic acids, organelles, orsignaling molecules from crossing the in situ-generated capturestructure. The in situ-generated capture structure may permit media topass through while not permitting a cell or a bead to cross the insitu-generated capture structure.

The process of introducing light activated polymerization can beperformed within the microfluidic device. Diffusion competes with thepolymerization process, so the ability to quickly create free radicalsmay be useful. Additionally, free radicals can quickly combine with freeoxygen. While photopolymerization is very efficient and quick in theabsence of oxygen in the media, when biological cells are present (thusrequiring the presence of oxygen), adjustments to the number ofinitiating radicals may be made to compensate. In fact, the limitingeffect of oxygen is helpful as chain termination happens more quicklyand limits the amount of extraneous polymer formed, particularly whenintroducing small limited amounts of polymer to form small capturestructures that do not entirely block entrance to or egress from a penor a channel.

In some embodiments, the step of initiating solidification of theflowable polymer may include optically illuminating the at least oneselected area of the flow region, and further where the step ofsolidification of the flowable polymer may include polymerizing polymersof the flowable polymer to form a solidified polymer network. The stepof introducing a flowable polymer may further include introducing aphotoactivatable polymerization initiator.

In some other embodiments, the step of initiating solidification of theflowable polymer may include changing a temperature at the at least oneselected area of the substrate. The step of solidification of thepolymer may further include gelling the polymer to form a polymernetwork. The step of changing the temperature at the selected area ofsubstrate may further include optically illuminating a thermal pad onthe substrate.

The in situ-generated capture structure can be formed by copolymerizingtwo polymers, one having, for example, an RGD peptide motif. In otherembodiments, a precursor pre-polymer (like prepolymer 401′ of FIG. 4D)may be modified to have such motif, and in situ-polymerization providesan in-situ generated capture structure including an assay analyte (e.g.,forming an in situ-generated capture structure 406C of FIG. 4D). Anotheralternative is to incorporate antibodies within a pre-polymer (like 407Bof FIG. 4C), and solidifying the polymer network in situ to provide insitu-generated capture structures already including antibody assayreagents (like 406B of FIG. 4C). Yet another alternative is to introducethe antibodies after the in situ-generated capture structure has beenformed (as in the conversion of in situ-generated capture structure 404to in situ-generated capture structure 406B of FIG. 4C). In one example,biotinylated or streptavidin sites can be introduced either throughoutthe solidified polymer network of the in situ-generated capturestructure or just on the surface, and streptavidin or biotin labeledantibodies may associate with respective binding pairs. Alternatively, amodified antibody may be devised, containing a photoactivatablefunctionality, such as benzophenone, which may be subjected tophotoinitiated insertion into the surface of the solidified polymernetwork of the in situ-generated capture structure at the same time, orafter formation of the in situ-generated capture structure, which wouldprovide a process similar to the conversion of in situ-generatedstructure 403, including a solidified polymer network, directly to insitu-generated capture structure 406B, where the solidified polymernetwork of the capture structure includes an assay reagent attached to afunctionalized site, of FIG. 4C (process not shown). The same types ofconversion strategies can be performed to equivalently introduce an insitu-generated capture structure including an assay analyte.

In one example of a process to introduce a polymer capture structurewithin a microfluidic device, a solution containing 10% w/v PEGDA (6 Kd)and 1% photoinitiator (IRGACURE 2959, 200 Da) may be flowed into themicrofluidic device. After allowing equilibration for less than 10 min,the desired region may be illuminated with UV light at approximately 340nm (+/−20 nm), having a power of 400 mW/cm², for 1 second, to initiatepolymerization creating an in situ-generated capture structure such asthat shown in the FIGS. 4-7.

A method is provided for preparing a microfluidic device including atleast a first in situ-generated capture structure, including: providingthe microfluidic device, where the microfluidic device comprises anenclosure including a substrate and microfluidic circuit materials,where the enclosure defines a flow region; introducing a first flowablefunctionalized pre-polymer into the flow region; and activatingsolidification of the first flowable functionalized pre-polymer at atleast one selected area of the enclosure, thereby forming the at least afirst in situ-generated capture structure therein. The step ofintroducing a first flowable functionalized pre-polymer may furtherinclude introducing a photoactivatable polymerization initiator into theflow region, where the step of introducing the photoactivatablepolymerization initiator may be performed before, concomitantly or afterthe step of introducing the first flowable pre-polymer. In someembodiments, the enclosure of the microfluidic device further includesat least one sequestration pen fluidically connected to the flow region,and the step of activating solidification includes activatingsolidification of the first flowable functionalized pre-polymer at atleast one selected area of the at least one sequestration pen. The atleast first in-situ generated capture structure may include a solidifiedpolymer network including one or more functionalized sites. The one ormore functionalized sites may include a biotin, avidin, or streptavidinmoiety. The one or more functionalized sites may be covalently bound toat least one component of the first flowable functionalized pre-polymer.Unsolidified flowable functionalized pre-polymer may be flowed out ofthe microfluidic device. In embodiments, where flowable functionalizedpre-polymer has been introduced to the least one sequestration pen, theunsolidified flowable functionalized pre-polymer may diffuse out of thepen, and then it may be flowed out of the microfluidic device

The method may further include flowing a first volume of a first fluidicmedium through the flow region of the microfluidic device, therebydiffusing unsolidified first flowable functionalized pre-polymer out ofthe at least one sequestration pen. The method may further includeintroducing a first functionalized assay reagent or assay analyte to theat least first capture structure within the enclosure, or alternativelywithin the at least one sequestration pen; and associating the firstfunctionalized assay reagent or assay analyte to the functionalizedsites of the solidified polymer network of the at least first capturestructure. The first functionalized assay reagent or assay analyte mayinclude an antibody, antigen, organic molecule, or an oligonucleotide.The organic molecule of the first functionalized assay reagent or assayanalyte may include a substrate to an enzyme, an antigen, a cell surfacemarker, a cytokine, or any suitable assay reagent or assay analytedescribed herein. The first functionalized assay reagent or assayanalyte may further include a moiety configured to associate the firstfunctionalized assay reagent or assay analyte with the functionalizedsite of the solidified polymer network of the at least first capturestructure. In various embodiments, the moiety configured to associatethe first functionalized assay reagent or assay analyte may include abiotin, avidin or streptavidin binding partner to the functionalizedsite of the solidified polymer network of the at least first capturestructure. In various embodiments, the first functionalized assayreagent or assay analyte may be a first assay reagent. The method mayfurther include flowing a second volume of the first fluidic mediumthrough the microfluidic device, thereby diffusing unassociated firstfunctionalized assay reagent or assay analyte out of the at least onesequestration pen. Once the unassociated functionalized assay reagent orassay analyte has diffused out of the pen, it may be flowed out of themicrofluidic device.

The method may further include a step of introducing a second or morefunctionalized assay reagent or assay analyte. The second or morefunctionalized assay reagent or assay analyte may associate with asecond or more functionalized sites of the solidified polymer network ofthe at least first capture structure. The second or more functionalizedassay reagent or assay analyte may be different from the firstfunctionalized assay reagent or assay analyte and/or detectablydifferentiable from the first functionalized assay reagent or assayanalyte. The second or more functionalized assay reagent or assayanalyte may be configured to be detected with a detection reagent thatis differentiable from the detection reagent that is used with the firstfunctionalized assay reagent or assay analyte. The second or morefunctionalized assay reagent or assay analyte may associate with asecond or more functionalized site on a second or more capture structurein the enclosure or, alternatively within the at least one sequestrationpen.

The method may further include a step of introducing a second or morecapture structure in the enclosure, or alternatively within the at leastone sequestration pen, where introducing the second or more capturestructure may include the steps of: introducing a further volume of thefirst fluidic medium into the flow region of the microfluidic device;introducing a second flowable functionalized pre-polymer into the flowregion; and activating solidification of the second flowablefunctionalized pre-polymer at at least a second selected area of theenclosure, or alternatively within the at least one sequestration pen,thereby forming the second in situ-generated capture structure therein;and flowing yet another volume of the first fluidic medium intro theflow region of the microfluidic device. The step of introducing thesecond flowable functionalized pre-polymer may further includeintroducing a photoactivatable polymerization initiator into the flowregion, where the step of introducing the photoactivatablepolymerization initiator may be performed before, concomitantly or afterthe step of introducing the second flowable pre-polymer. After thesecond capture structure is formed, the second functionalized assayreagent or assay analyte may be flowed in, similarly to the firstfunctionalized assay reagent or assay analyte and allowed to associatewith the second capture structure. After association of the secondfunctionalized assay reagent and assay analyte is complete, excessunassociated functionalized assay reagent or assay analyte may bediffused out of the sequestration pen and, optionally, flowed out of themicrofluidic device.

The method may further include a step of introducing a third or morecapture structure into the at least one sequestration pen, whereintroducing the third or more capture structure may include: introducinga further volume of the first fluidic medium into the flow region of themicrofluidic device; introducing a third flowable functionalizedpre-polymer into the flow region; and activating solidification of thethird flowable functionalized pre-polymer at at least a third selectedarea of the enclosure or, alternatively, within the at least onesequestration pen, thereby forming the third in situ-generated capturestructure therein; and flowing yet another volume of the first fluidicmedium intro the flow region of the microfluidic device. The step ofintroducing a third flowable functionalized pre-polymer may furtherinclude introducing a photoactivatable polymerization initiator into theflow region, where the step of introducing the photoactivatablepolymerization initiator may be performed before, concomitantly or afterthe step of introducing the third flowable pre-polymer. The thirdfunctionalized assay reagent or assay analyte may be introduced in asimilar manner to the third capture structure, as described above forthe first and/or the second functionalized assay reagent or assayanalyte. The third functionalized assay reagent or assay analyte may bedifferent from the first or second functionalized assay reagent, and/ormay be detectably distinguishable from the first or the secondfunctionalized assay reagent or assay analyte.

In some embodiments, the first flowable functionalized pre-polymer maybe different from the second flowable functionalized pre-polymer. Inother embodiments, the first flowable functionalized pre-polymer may bethe same as the second flowable functionalized pre-polymer. In variousembodiments, each of the first, second, and third flowablefunctionalized pre-polymer may be different from each other. In otherembodiments, each of the first, second, and third flowablefunctionalized pre-polymer may be the same functionalized pre-polymer.In various embodiments of the methods, the solidified polymer network ofany of the first, second or third in situ-generated capture structuresmay include a synthetic polymer, a modified synthetic polymer, or abiological polymer. In some embodiments, the synthetic polymermodifications comprise size modification motifs, cleavage motifs,reactive terminal moieties, and/or cell recognition motifs. Thesolidified polymer network may include at least one of a polyethyleneglycol (PEG), modified polyethylene glycol, polylactic acid (PLA),modified polylactic acid, polyglycolic acid (PGA), modified polyglycolicacid, polyacrylamide (PAM), modified polyacrylamide,poly-N-isopropylacrylamide (PNIPAm), modifiedpoly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinylalcohol, polyacrylic acid (PAA), modified polyacrylic acid,polycaprolactone (PCL), modified polycaprolactone, fibronectin, modifiedfibronectin, collagen, modified collagen, laminin, modified laminin,polysaccharide, modified polysaccharide, or a co-polymer in anycombination. In yet other embodiments, the solidified polymer networkmay include at least one of a polyethylene glycol, modified polyethyleneglycol, polylactic acid (PLA), modified polylactic acid, polyglycolicacid (PGA), modified polyglycolic acid, polyvinyl alcohol (PVA),modified polyvinyl alcohol, polyacrylic acid (PAA), modified polyacrylicacid, fibronectin, modified fibronectin, collagen, modified collagen,laminin, modified laminin, or a co-polymer in any combination. In someembodiments, the polymer of the solidified polymer network may be amodified PEG polymer. The polymer may be a star, 4-arm or 2-arm PEGdiacrylate polymer.

Kits.

In yet another aspect, a kit is provided, including: a microfluidicdevice having an enclosure including a substrate, microfluidic circuitmaterial, and, optionally, a cover, where the enclosure defines a flowregion; and a functionalized pre-polymer that can be controllablyactivated to form a solidified polymer network. The kit can furtherinclude an assay reagent or an assay analyte, which may be part of thefunctionalized pre-polymer, mixed with the functionalized pre-polymer,or provided separately from the functionalized pre-polymer (e.g., in aseparate vial, tube, etc.). Alternatively, a kit is provided including:a microfluidic device having an enclosure including a substrate,microfluidic circuit material, and, optionally, a cover, where theenclosure defines a flow region; and at least one in situ-generatedcapture structure disposed within the enclosure, wherein the at leastone in situ-generated capture structure includes a solidified polymernetwork (e.g., microfluidic device 400, 700). The kit can furtherinclude an assay reagent, which may be integral to or associated withthe in situ-generated capture structure or which may be providedseparately (e.g., in a vial, tube, etc.). The microfluidic device ineither kit can include at least one sequestration pen within theenclosure. For kits in which the in situ-generated capture structure isalready disposed within the microfluidic device, the in situ-generatedcapture structure can be located within the flow region, a sequestrationpen of the microfluidic device (e.g., an isolation region within thesequestration pen), or both The solidified polymer network may furtherinclude one or more functionalized sites. The functionalized sites ofthe solidified polymer network may be any functionalized sites asdescribed herein, and may include biotin, avidin, streptavidin, or anycombination thereof.

In various embodiments of the kit including a microfluidic deviceincluding at least one in situ-generated capture structure, thesolidified polymer network may include a synthetic polymer, a modifiedsynthetic polymer, or a biological polymer. In embodiments of the kitswherein a functionalized pre-polymer is provided, the functionalizedpre-polymer may include a synthetic polymer, a modified syntheticpolymer, or a biological polymer. In some embodiments, the syntheticpolymer modifications comprise size modification motifs, cleavagemotifs, reactive terminal moieties, and/or cell recognition motifs. Thesolidified polymer network or functionalized pre-polymer may include atleast one of a polyethylene glycol (PEG), modified polyethylene glycol,polylactic acid (PLA), modified polylactic acid, polyglycolic acid(PGA), modified polyglycolic acid, polyacrylamide (PAM), modifiedpolyacrylamide, poly-N-isopropylacrylamide (PNIPAm), modifiedpoly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinylalcohol, polyacrylic acid (PAA), modified polyacrylic acid,polycaprolactone (PCL), modified polycaprolactone, fibronectin, modifiedfibronectin, collagen, modified collagen, laminin, modified laminin,polysaccharide, modified polysaccharide, or a co-polymer in anycombination. In yet other embodiments, the solidified polymer network orthe functionalized pre-polymer may include at least one of apolyethylene glycol, modified polyethylene glycol, polylactic acid(PLA), modified polylactic acid, polyglycolic acid (PGA), modifiedpolyglycolic acid, polyvinyl alcohol (PVA), modified polyvinyl alcohol,polyacrylic acid (PAA), modified polyacrylic acid, fibronectin, modifiedfibronectin, collagen, modified collagen, laminin, modified laminin, ora co-polymer in any combination. In some embodiments, the polymer of thesolidified polymer network or of the functionalized pre-polymer may be amodified PEG polymer. The polymer may be a star, 4-arm or 2-arm PEGdiacrylate polymer. In various embodiments, wherein the enclosure or atleast one sequestration pen includes more than one in situ-generatedcapture structure, each of the capture structures include the samepolymer, or may alternatively include polymers that are different foreach of the first in situ-generated capture structure, the second insitu-generated capture structure, the third in situ-generated capturestructure, and so on. In kits providing a functionalized pre-polymer,more than one functionalized prepolymer may be provided. The kit mayinclude more than one polymer, which can be combined together with theat least first functionalized polymer to form a flowable polymersolution configured to be solidified to form a solidified polymernetwork. The at least first functionalized polymer may be provided as aflowable polymer solution or may be provided as a gel, dehydrated, orlyophilized form, any of which may be configured to be formulated by theend user as a flowable polymer solution by dilution with a fluidicmedium. Other polymers provided in the kit, which may be used to formthe in situ-generated capture structure, may be provided in one or moreseparate containers from the at least first functionalized polymer.

The assay reagent or assay analyte of the kit may be any assay reagentor assay analyte described herein. The assay reagent or assay analytemay include a functional moiety configured to associate with thefunctionalized sites of the solidified polymer network of the at leastone capture structure. The assay reagent or assay analyte may beprovided in a formulation configured to be ready to introduce into theflow region of the microfluidic device. Alternatively, the assay reagentor assay analyte may be provided in a solid or lyophilized form, withinstructions for dissolution into an appropriate medium for introductioninto the microfluidic device and subsequent association with thefunctionalized sites of the solidified polymer network of the insitu-generated capture structure. In some embodiments of the kits, morethan one assay reagent or more than one assay analyte, in any suitablecombination, may be provided for multiplex experiments, and may any ofthe assay reagents or analytes as described in the following paragraphs.

In various embodiments of the kits including a microfluidic deviceincluding at least one in situ-generated capture structure, thesolidified polymer network of the in situ-generated capture structuremay already include an assay reagent or assay analyte (e.g.,microfluidic device 450, where the assay reagent or assay analyte isalready present within/on the solidified polymer network of the at leastone capture structure of the microfluidic device as supplied). The assayreagent or assay analyte may be covalently or non-covalently bound tothe one or more functionalized sites of the solidified polymer network.In some embodiments, the assay reagent or assay analyte may benon-covalently bound to the one or more functionalized sites of thesolidified polymer network via a biotin/streptavidin or biotin/avidincomplex.

Whether the assay reagent or assay analyte is provided alreadyincorporated as part of the solidified polymer network of the insitu-generated capture structure or as a component of a kit to preparean in situ-generated capture structure having such assay reagent orassay analyte, the assay reagent or assay analyte may be any suitablemoiety as described herein. The assay reagent or assay analyte,incorporated or to be incorporated within an in situ-generated capturestructure, may be a protein, a nucleic acid, an organic molecule, or asaccharide. In some embodiments of the kit including a microfluidicdevice including at least one in situ-generated capture structure, theassay reagent or assay analyte may be an antibody, and may be any kindof antibody as described herein. In other embodiments, the assay reagentor assay analyte, incorporated or to be incorporated within an insitu-generated capture structures may be an antigen. The assay reagentor assay analyte that is an antigen may be any suitable antigen asdescribed herein. In yet other embodiments, the assay reagent,incorporated or to be incorporated within an in situ-generated capturestructure may be is an oligonucleotide. The oligonucleotide assayreagent may be any suitable oligonucleotide as described herein.

In some embodiments of the kits, the assay reagent or assay analyte mayinclude a detectable label. The detectable label of the assay reagent orassay analyte may be a fluorescent, colorimetric, or luminescent label.In some embodiments, when the assay reagent or assay analyte includes adetectable label, the label is not detectable until the assay process isunderway, and the detectable label is generated or liberated from theassay reagent or assay analyte.

In other embodiments of the kits, the kit may further include adetection reagent. The detection reagent may include a detectable label.The detectable label of the detection reagent may include a fluorescent,colorimetric, or luminescent label. In some embodiments, the detectablelabel of the detection reagent may be fluorescent. In some embodiments,the detection reagent includes at least a first antibody. In someembodiments, the detection reagent may include a second antibody, wherethe second antibody is a secondary antibody to the assay process andincorporates the detectable label for the combination of the first andsecond antibody that comprises the detection reagent. In otherembodiments, the detection reagent may include an intercalating dye. Inyet other embodiments, the detection reagent may include a FRET labeledoligonucleotide, which may be any FRET labeled oligonucleotide asdescribed herein.

In various embodiments of the kits, the more than one detection reagentmay be provided. A first detection reagent of the more than onedetection reagent may be spectrally distinct from a second detectionreagent, and so on for each different assay reagent or assay analyte.

In other embodiments of the kits, the microfluidic device may includetwo or more capture structures disposed within the enclosure, or,alternatively, within a sequestration pen therein, where a firstsolidified polymer network of a first capture structure alreadyincludes/is designed to incorporate a first assay reagent or assayanalyte and a second solidified polymer network of a second capturestructure already includes/or is designed to incorporate a second assayreagent or assay analyte, and so on for each additional capturestructure in the enclosure or, alternatively, the at least onesequestration pen. The first assay reagent or assay analyte may bedifferent from the second assay reagent or assay analyte, and so on foreach additional assay reagent or assay analyte incorporated or designedto be incorporated within each additional capture structure in the atleast one sequestration pen. The first capture structure and the secondcapture structure may be disposed in different locations within theenclosure, or at least one sequestration pen therein, of themicrofluidic device.

When a first capture structure incorporates or is designed toincorporate a first assay reagent or assay analyte, and a second capturestructure incorporates or is designed to incorporate a second assayreagent or assay analyte, the kit may further include a respective firstdetection reagent and a second detection reagent, where the firstdetection reagent may be different from the second detection reagent.The first detection reagent and the second detection reagent, and so on,for any additional assay reagents or analytes incorporated or configuredto be incorporated on capture structures, may include any detectionreagent as described herein, and may be selected independently. In someembodiments, the first detection reagent may include at least a firstprimary antibody and the second detection reagent comprises at least asecond primary antibody directed to the respective biological targets ofeach assay. Each detection reagent including a primary antibody mayfurther include a secondary antibody, which itself may include thedetectable label for each assay being performed. When more than onecapture structure is provided in the enclosure, or alternatively in theat least one sequestration pen therein, either each of the labels of therespective detection reagents are spectrally distinct or the labels ofthe respective detection reagents are spatially distinct. In someembodiments, the labels are both spectrally and spatially distinct.

In various embodiments of the kit including a microfluidic deviceincluding at least one in situ-generated capture structure, themicrofluidic device may further include a plurality of sequestrationpens. In some embodiments, each of the plurality of sequestration pensmay include at least one capture structure comprising a solidifiedpolymer network. The plurality of sequestration pens may be configuredas described for any sequestration pen described herein and in anycombination. The microfluidic device of the kit may further include anycomponent or feature of any of microfluidic devices 100, 200, 23, 250,280, 290, 320, 400, 450, 500, 700 as described herein, in anycombination.

In some embodiments of the kits, one or more fluidic media may beincluded, and may further include one or more additives described hereinto provide enhanced growth, viability or portability, includingadditives for a dynamic coating within the microfluidic device. In otherembodiments of the kit, one or more of the surfaces of the enclosure mayinclude a coating. The coating may be any coating as described herein.In some embodiments, the coating is a covalent coating that provides aconditioned surface. The covalent coating may be present on all theinterior surfaces of the enclosure of the microfluidic device. In someembodiments, the covalent coating providing a conditioned surface may behydrophilic.

In various embodiments of the kits, the kit may further include aphotoactivatable polymerization initiator. The photoactivatablepolymerization initiator may be provided in a separate container fromthe fluidic medium and/or functionalized pre-polymer(s).

Example 1. Hydrogel Cytokine Assay

T Cells.

CD3+ cells from AllCells Inc. and mixed with anti-CD3/anti-CD28 magneticbeads (Dynabeads®, ThermoFisher Scientific, Cat. No. 11453D) at a ratioof 1 bead/1 cell. The mixture was incubated in the same medium as theculturing experiment itself, for 48 hours in a 5% CO₂ incubator at 37°C. Following the incubation, the T cell/bead mixture was resuspended foruse.

Culture Medium.

RPMI-1640 (GIBCO®, ThermoFisher Scientific, Cat. No. 11875-127), 10%FBS, 2% Human AB serum (50 U/ml IL2; R&D Systems).

Priming Procedure:

250 microliters of 100% carbon dioxide was flowed in at a rate of 12microliters/sec. This was followed by 250 microliters of PBS containing0.1% Pluronic® F27 (Life Technologies® Cat# P6866), flowed in at 12microliters/sec. The final step of priming included 250 microliters ofPBS, flowed in at 12 microliters/sec. Introduction of the culture mediumfollows.

Perfusion Regime (During Cell Culturing on Chip:

The perfusion method was either of the following two methods:

1. Perfuse at 0.01 microliters/sec for 2 h; perfuse at 2 microliters/secfor 64 sec; and repeat.

2. Perfuse at 0.02 microliters/sec for 100 sec; stop flow 500 sec;perfuse at 2 microliters/sec for 64 sec; and repeat.

System and Microfluidic Device: System and Microfluidic Device:

Manufactured by Berkeley Lights, Inc. The system included at least aflow controller, temperature controller, fluidic medium conditioning andpump component, light source for light activated DEP configurations,microfluidic device, mounting stage, and a camera. The sequestrationpens have a volume of about 7×10⁵ cubic microns.

Hydrogel Preparation.

Streptavidin amine (Nanocs) in N-morpholino ethanesulfonic acid (MES)was diluted in 0.1M carbonate-bicarbonate (CBB buffer at pH 9.0) andreacted with Ac-PEG-hydroxysuccinimide in a ratio calculated to providea 5% wt percentage for a final concentration of 10 mg/200 microliter.The reaction was continued at least overnight at 4° C.

A solution of Igracure photoinitiator at 0.606 wt % solution indeionized ultra filtered water (DIUF) was made.

The prepolymer for the functional hydrogel was made by solubilizing a 5wt % solution of Ac PEG star solid (4 arm PEG acrylate (10 k MW) fromLaysan Bio (#4arm-PEG-ACRYL-10 k-1 g)) in the 0.606% photoinitiatorsolution.

A 160 microliter prepolymer lot was made by combining 28 microliter ofthe NHS-PEG-streptavidin conjugate and 132 microliters of the Ac PEGstar acrylate/photoinitiator solution.

A primed microfluidic device was loaded with the prepolymer solution at0.5 microliter/sec, and incubated for at least 1 hour prior tophotoinitiation to allow diffusion of prepolymer into pens of themicrofluidic device. After incubation was complete, a 10 sec exposure tolight was used to initiate polymer solidification at the bottom cornerof pens.

After solidification was initiated, a set of rinses were used to removeexcess soluble polymers and initiator, including 2×250 microliters PBSat 8 microliters/sec; 250 microliters PBS at 0.2 microliters/sec; and anovernight rinse in PBS (250 microliters at 0.005 microliters/sec).

Hydrogel Functionalization.

The hydrogel prepared microfluidic device was loaded with 1 microgram/mLcapture antibody (Biotinylated goat anti-human TNF alpha from R&DSystems (#BAF210) where the in situ-generated capture antibody solutionwas flowed in within 250 microliters at 5 microliters/sec, followed by asecond flow period of 250 microliters of the in situ-generated captureantibody solution at 0.075 microliters/sec. After completing theintroduction of the in situ-generated capture antibody, the microfluidicdevice was flushed with PBS (250 microliters at 5 microliters/sec)×5.

T-Cell Introduction and Detection of TNF Alpha.

T-cells were introduced and cultured for overnight at 37° C. A firstdetection antibody was introduced after the end of the incubationperiod, by flowing a solution of Rabbit anti-human TNF alpha from Abcam(#ab9635), within 250 microliters at 5 microliters/sec, followed by asecond flow period of 250 microliters of the first detection antibodysolution at 0.075 microliters/sec. After completing the introduction ofthe first detection antibody, the microfluidic device was flushed withPBS (250 microliters at 5 microliters/sec)×5.

A secondary detection antibody (Alexa 488 goat anti-rabbit IgG from LifeTechnologies (#A11053)) was then introduced at a concentration of 2micrograms/ml by flowing 250 microliters of the solution at 5microliters/sec; followed by a second 250 microliter flow of the 2microgram/ml solution at 0.075 microliter/sec.

FIGS. 6A-C showed the ability to differentiate between highly secretingT cells, moderately secreting T cell and poorly secreting T cells. InFIG. 6A, fluorescence is just detectable for this pen containing poorlysecreting T cell(s). The left hand image is brightfield, and four cellsmay be seen, while the right hand image shows the fluorescence image ofthe same pen, where the functionalized hydrogel is faintly visible atthe lower left corner of the pen.

FIG. 6B shows a moderately secreting set of T cells. The left hand imageis that of one to three T cells in a different pen, and the right handfluorescence image of that same pen clearly shows significantfluorescence concentrated at the surface of the functionalized hydrogelin the lower left corner of the pen.

FIG. 6C shows a highly secreting set of T cells in a third pen. The lefthand image in FIG. 6C is brightfield and shows a group of about 4-5 Tcells in a clump as well as one solo cell. The right hand image showsthe same third pen under fluorescent detection where both thefunctionalized hydrogel is well illuminated as well as the clump ofcells in the pen.

This example clearly demonstrated that differing levels of TNF alphacytokine production can be detected and ranked within the microfluidicpens.

Microfluidic Devices and Systems for Operating and Observing SuchDevices.

FIG. 1A illustrates an example of a microfluidic device 100 and a system150 which can be used for generation of embryos in vitro, includingselecting and evaluating ova and/or oocytes and/or sperm. A perspectiveview of the microfluidic device 100 is shown having a partial cut-awayof its cover 110 to provide a partial view into the microfluidic device100. The microfluidic device 100 generally comprises a microfluidiccircuit 120 comprising a flow path 106 through which a fluidic medium180 can flow, optionally carrying one or more micro-objects (not shown)into and/or through the microfluidic circuit 120. Although a singlemicrofluidic circuit 120 is illustrated in FIG. 1A, suitablemicrofluidic devices can include a plurality (e.g., 2 or 3) of suchmicrofluidic circuits. Regardless, the microfluidic device 100 can beconfigured to be a nanofluidic device. As illustrated in FIG. 1A, themicrofluidic circuit 120 may include a plurality of microfluidicsequestration pens 124, 126, 128, and 130, where each sequestration pensmay have one or more openings in fluidic communication with flow path106. In some embodiments of the device of FIG. 1A, the sequestrationpens may have only a single opening in fluidic communication with theflow path 106. As discussed further below, the microfluidicsequestration pens comprise various features and structures that havebeen optimized for retaining micro-objects in the microfluidic device,such as microfluidic device 100, even when a medium 180 is flowingthrough the flow path 106. Before turning to the foregoing, however, abrief description of microfluidic device 100 and system 150 is provided.

As generally illustrated in FIG. 1A, the microfluidic circuit 120 isdefined by an enclosure 102. Although the enclosure 102 can bephysically structured in different configurations, in the example shownin FIG. 1A the enclosure 102 is depicted as comprising a supportstructure 104 (e.g., a base), a microfluidic circuit structure 108, anda cover 110. The support structure 104, microfluidic circuit structure108, and cover 110 can be attached to each other. For example, themicrofluidic circuit structure 108 can be disposed on an inner surface109 of the support structure 104, and the cover 110 can be disposed overthe microfluidic circuit structure 108. Together with the supportstructure 104 and cover 110, the microfluidic circuit structure 108 candefine the elements of the microfluidic circuit 120.

The support structure 104 can be at the bottom and the cover 110 at thetop of the microfluidic circuit 120 as illustrated in FIG. 1A.Alternatively, the support structure 104 and the cover 110 can beconfigured in other orientations. For example, the support structure 104can be at the top and the cover 110 at the bottom of the microfluidiccircuit 120. Regardless, there can be one or more ports 107 eachcomprising a passage into or out of the enclosure 102. Examples of apassage include a valve, a gate, a pass-through hole, or the like. Asillustrated, port 107 is a pass-through hole created by a gap in themicrofluidic circuit structure 108. However, the port 107 can besituated in other components of the enclosure 102, such as the cover110. Only one port 107 is illustrated in FIG. 1A but the microfluidiccircuit 120 can have two or more ports 107. For example, there can be afirst port 107 that functions as an inlet for fluid entering themicrofluidic circuit 120, and there can be a second port 107 thatfunctions as an outlet for fluid exiting the microfluidic circuit 120.Whether a port 107 function as an inlet or an outlet can depend upon thedirection that fluid flows through flow path 106.

The support structure 104 can comprise one or more electrodes (notshown) and a substrate or a plurality of interconnected substrates. Forexample, the support structure 104 can comprise one or moresemiconductor substrates, each of which is electrically connected to anelectrode (e.g., all or a subset of the semiconductor substrates can beelectrically connected to a single electrode). The support structure 104can further comprise a printed circuit board assembly (“PCBA”). Forexample, the semiconductor substrate(s) can be mounted on a PCBA.

The microfluidic circuit structure 108 can define circuit elements ofthe microfluidic circuit 120. Such circuit elements can comprise spacesor regions that can be fluidly interconnected when microfluidic circuit120 is filled with fluid, such as flow regions (which may include or beone or more flow channels), chambers, pens, traps, and the like. In themicrofluidic circuit 120 illustrated in FIG. 1A, the microfluidiccircuit structure 108 comprises a frame 114 and a microfluidic circuitmaterial 116. The frame 114 can partially or completely enclose themicrofluidic circuit material 116. The frame 114 can be, for example, arelatively rigid structure substantially surrounding the microfluidiccircuit material 116. For example, the frame 114 can comprise a metalmaterial.

The microfluidic circuit material 116 can be patterned with cavities orthe like to define circuit elements and interconnections of themicrofluidic circuit 120. The microfluidic circuit material 116 cancomprise a flexible material, such as a flexible polymer (e.g. rubber,plastic, elastomer, silicone, polydimethylsiloxane (“PDMS”), or thelike), which can be gas permeable. Other examples of materials that cancompose microfluidic circuit material 116 include molded glass, anetchable material such as silicone (e.g. photo-patternable silicone or“PPS”), photo-resist (e.g., SU8), or the like. In some embodiments, suchmaterials—and thus the microfluidic circuit material 116—can be rigidand/or substantially impermeable to gas. Regardless, microfluidiccircuit material 116 can be disposed on the support structure 104 andinside the frame 114.

The cover 110 can be an integral part of the frame 114 and/or themicrofluidic circuit material 116. Alternatively, the cover 110 can be astructurally distinct element, as illustrated in FIG. 1A. The cover 110can comprise the same or different materials than the frame 114 and/orthe microfluidic circuit material 116. Similarly, the support structure104 can be a separate structure from the frame 114 or microfluidiccircuit material 116 as illustrated, or an integral part of the frame114 or microfluidic circuit material 116. Likewise, the frame 114 andmicrofluidic circuit material 116 can be separate structures as shown inFIG. 1A or integral portions of the same structure.

In some embodiments, the cover 110 can comprise a rigid material. Therigid material may be glass or a material with similar properties. Insome embodiments, the cover 110 can comprise a deformable material. Thedeformable material can be a polymer, such as PDMS. In some embodiments,the cover 110 can comprise both rigid and deformable materials. Forexample, one or more portions of cover 110 (e.g., one or more portionspositioned over sequestration pens 124, 126, 128, 130) can comprise adeformable material that interfaces with rigid materials of the cover110. In some embodiments, the cover 110 can further include one or moreelectrodes. The one or more electrodes can comprise a conductive oxide,such as indium-tin-oxide (ITO), which may be coated on glass or asimilarly insulating material. Alternatively, the one or more electrodescan be flexible electrodes, such as single-walled nanotubes,multi-walled nanotubes, nanowires, clusters of electrically conductivenanoparticles, or combinations thereof, embedded in a deformablematerial, such as a polymer (e.g., PDMS). Flexible electrodes that canbe used in microfluidic devices have been described, for example, inU.S. 2012/0325665 (Chiou et al.), the contents of which are incorporatedherein by reference. In some embodiments, the cover 110 can be modified(e.g., by conditioning all or part of a surface that faces inward towardthe microfluidic circuit 120) to support cell adhesion, viability and/orgrowth. The modification may include a coating of a synthetic or naturalpolymer. In some embodiments, the cover 110 and/or the support structure104 can be transparent to light. The cover 110 may also include at leastone material that is gas permeable (e.g., PDMS or PPS).

FIG. 1A also shows a system 150 for operating and controllingmicrofluidic devices, such as microfluidic device 100. System 150includes an electrical power source 192, an imaging device 194(incorporated within imaging module 164, where device 194 is notillustrated in FIG. 1A, per se), and a tilting device 190 (part oftilting module 166, where device 190 is not illustrated in FIG. 1A).

The electrical power source 192 can provide electric power to themicrofluidic device 100 and/or tilting device 190, providing biasingvoltages or currents as needed. The electrical power source 192 can, forexample, comprise one or more alternating current (AC) and/or directcurrent (DC) voltage or current sources. The imaging device 194 (part ofimaging module 164, discussed below) can comprise a device, such as adigital camera, for capturing images inside microfluidic circuit 120. Insome instances, the imaging device 194 further comprises a detectorhaving a fast frame rate and/or high sensitivity (e.g. for low lightapplications). The imaging device 194 can also include a mechanism fordirecting stimulating radiation and/or light beams into the microfluidiccircuit 120 and collecting radiation and/or light beams reflected oremitted from the microfluidic circuit 120 (or micro-objects containedtherein). The emitted light beams may be in the visible spectrum andmay, e.g., include fluorescent emissions. The reflected light beams mayinclude reflected emissions originating from an LED or a wide spectrumlamp, such as a mercury lamp (e.g. a high pressure mercury lamp) or aXenon arc lamp. As discussed with respect to FIG. 3B, the imaging device194 may further include a microscope (or an optical train), which may ormay not include an eyepiece.

System 150 further comprises a tilting device 190 (part of tiltingmodule 166, discussed below) configured to rotate a microfluidic device100 about one or more axes of rotation. In some embodiments, the tiltingdevice 190 is configured to support and/or hold the enclosure 102comprising the microfluidic circuit 120 about at least one axis suchthat the microfluidic device 100 (and thus the microfluidic circuit 120)can be held in a level orientation (i.e. at 0° relative to x- andy-axes), a vertical orientation (i.e. at 90° relative to the x-axisand/or the y-axis), or any orientation therebetween. The orientation ofthe microfluidic device 100 (and the microfluidic circuit 120) relativeto an axis is referred to herein as the “tilt” of the microfluidicdevice 100 (and the microfluidic circuit 120). For example, the tiltingdevice 190 can tilt the microfluidic device 100 at 0.1°, 02°, 0.3°,0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 10°, 15°, 20°,25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 90° relativeto the x-axis or any degree therebetween. The level orientation (andthus the x- and y-axes) is defined as normal to a vertical axis definedby the force of gravity. The tilting device can also tilt themicrofluidic device 100 (and the microfluidic circuit 120) to any degreegreater than 90° relative to the x-axis and/or y-axis, or tilt themicrofluidic device 100 (and the microfluidic circuit 120) 180° relativeto the x-axis or the y-axis in order to fully invert the microfluidicdevice 100 (and the microfluidic circuit 120). Similarly, in someembodiments, the tilting device 190 tilts the microfluidic device 100(and the microfluidic circuit 120) about an axis of rotation defined byflow path 106 or some other portion of microfluidic circuit 120.

In some instances, the microfluidic device 100 is tilted into a verticalorientation such that the flow path 106 is positioned above or below oneor more sequestration pens. The term “above” as used herein denotes thatthe flow path 106 is positioned higher than the one or moresequestration pens on a vertical axis defined by the force of gravity(i.e. an object in a sequestration pen above a flow path 106 would havea higher gravitational potential energy than an object in the flowpath). The term “below” as used herein denotes that the flow path 106 ispositioned lower than the one or more sequestration pens on a verticalaxis defined by the force of gravity (i.e. an object in a sequestrationpen below a flow path 106 would have a lower gravitational potentialenergy than an object in the flow path).

In some instances, the tilting device 190 tilts the microfluidic device100 about an axis that is parallel to the flow path 106. Moreover, themicrofluidic device 100 can be tilted to an angle of less than 90° suchthat the flow path 106 is located above or below one or moresequestration pens without being located directly above or below thesequestration pens. In other instances, the tilting device 190 tilts themicrofluidic device 100 about an axis perpendicular to the flow path106. In still other instances, the tilting device 190 tilts themicrofluidic device 100 about an axis that is neither parallel norperpendicular to the flow path 106.

System 150 can further include a media source 178. The media source 178(e.g., a container, reservoir, or the like) can comprise multiplesections or containers, each for holding a different fluidic medium 180.Thus, the media source 178 can be a device that is outside of andseparate from the microfluidic device 100, as illustrated in FIG. 1A.Alternatively, the media source 178 can be located in whole or in partinside the enclosure 102 of the microfluidic device 100. For example,the media source 178 can comprise reservoirs that are part of themicrofluidic device 100.

FIG. 1A also illustrates simplified block diagram depictions of examplesof control and monitoring equipment 152 that constitute part of system150 and can be utilized in conjunction with a microfluidic device 100.As shown, examples of such control and monitoring equipment 152 includea master controller 154 comprising a media module 160 for controllingthe media source 178, a motive module 162 for controlling movementand/or selection of micro-objects (not shown) and/or medium (e.g.,droplets of medium) in the microfluidic circuit 120, an imaging module164 for controlling an imaging device 194 (e.g., a camera, microscope,light source or any combination thereof) for capturing images (e.g.,digital images), and a tilting module 166 for controlling a tiltingdevice 190. The control equipment 152 can also include other modules 168for controlling, monitoring, or performing other functions with respectto the microfluidic device 100. As shown, the equipment 152 can furtherinclude a display device 170 and an input/output device 172.

The master controller 154 can comprise a control module 156 and adigital memory 158. The control module 156 can comprise, for example, adigital processor configured to operate in accordance with machineexecutable instructions (e.g., software, firmware, source code, or thelike) stored as non-transitory data or signals in the memory 158.Alternatively, or in addition, the control module 156 can comprisehardwired digital circuitry and/or analog circuitry. The media module160, motive module 162, imaging module 164, tilting module 166, and/orother modules 168 can be similarly configured. Thus, functions,processes acts, actions, or steps of a process discussed herein as beingperformed with respect to the microfluidic device 100 or any othermicrofluidic apparatus can be performed by any one or more of the mastercontroller 154, media module 160, motive module 162, imaging module 164,tilting module 166, and/or other modules 168 configured as discussedabove. Similarly, the master controller 154, media module 160, motivemodule 162, imaging module 164, tilting module 166, and/or other modules168 may be communicatively coupled to transmit and receive data used inany function, process, act, action or step discussed herein.

The media module 160 controls the media source 178. For example, themedia module 160 can control the media source 178 to input a selectedfluidic medium 180 into the enclosure 102 (e.g., through an inlet port107). The media module 160 can also control removal of media from theenclosure 102 (e.g., through an outlet port (not shown)). One or moremedia can thus be selectively input into and removed from themicrofluidic circuit 120. The media module 160 can also control the flowof fluidic medium 180 in the flow path 106 inside the microfluidiccircuit 120. For example, in some embodiments media module 160 stops theflow of media 180 in the flow path 106 and through the enclosure 102prior to the tilting module 166 causing the tilting device 190 to tiltthe microfluidic device 100 to a desired angle of incline.

The motive module 162 can be configured to control selection, trapping,and movement of micro-objects (not shown) in the microfluidic circuit120. As discussed below with respect to FIGS. 1B and 1C, the enclosure102 can comprise a dielectrophoresis (DEP), optoelectronic tweezers(OET) and/or opto-electrowetting (OEW) configuration (not shown in FIG.1A), and the motive module 162 can control the activation of electrodesand/or transistors (e.g., phototransistors) to select and movemicro-objects (not shown) and/or droplets of medium (not shown) in theflow path 106 and/or sequestration pens 124, 126, 128, 130.

The imaging module 164 can control the imaging device 194. For example,the imaging module 164 can receive and process image data from theimaging device 194. Image data from the imaging device 194 can compriseany type of information captured by the imaging device 194 (e.g., thepresence or absence of micro-objects, droplets of medium, accumulationof label, such as fluorescent label, etc.). Using the informationcaptured by the imaging device 194, the imaging module 164 can furthercalculate the position of objects (e.g., micro-objects, droplets ofmedium) and/or the rate of motion of such objects within themicrofluidic device 100.

The tilting module 166 can control the tilting motions of tilting device190. Alternatively, or in addition, the tilting module 166 can controlthe tilting rate and timing to optimize transfer of micro-objects to theone or more sequestration pens via gravitational forces. The tiltingmodule 166 is communicatively coupled with the imaging module 164 toreceive data describing the motion of micro-objects and/or droplets ofmedium in the microfluidic circuit 120. Using this data, the tiltingmodule 166 may adjust the tilt of the microfluidic circuit 120 in orderto adjust the rate at which micro-objects and/or droplets of medium movein the microfluidic circuit 120. The tilting module 166 may also usethis data to iteratively adjust the position of a micro-object and/ordroplet of medium in the microfluidic circuit 120.

In the example shown in FIG. 1A, the microfluidic circuit 120 isillustrated as comprising a microfluidic channel 122 and sequestrationpens 124, 126, 128, 130. Each pen comprises an opening to channel 122,but otherwise is enclosed such that the pens can substantially isolatemicro-objects inside the pen from fluidic medium 180 and/ormicro-objects in the flow path 106 of channel 122 or in other pens. Thewalls of the sequestration pen extend from the inner surface 109 of thebase to the inside surface of the cover 110 to provide enclosure. Theopening of the pen to the microfluidic channel 122 is oriented at anangle to the flow 106 of fluidic medium 180 such that flow 106 is notdirected into the pens. The flow may be tangential or orthogonal to theplane of the opening of the pen. In some instances, pens 124, 126, 128,130 are configured to physically corral one or more micro-objects withinthe microfluidic circuit 120. Sequestration pens in accordance with thepresent disclosure can comprise various shapes, surfaces and featuresthat are optimized for use with DEP, OET, OEW, fluid flow, and/orgravitational forces, as will be discussed and shown in detail below.

The microfluidic circuit 120 may comprise any number of microfluidicsequestration pens. Although five sequestration pens are shown,microfluidic circuit 120 may have fewer or more sequestration pens. Asshown, microfluidic sequestration pens 124, 126, 128, and 130 ofmicrofluidic circuit 120 each comprise differing features and shapeswhich may provide one or more benefits useful in producing an embryo,such as isolating one ovum from an adjacent ovum. Testing, stimulatingand fertilizing may all be performed on an individual basis and, in someembodiments, may be performed on an individual time scale. In someembodiments, the microfluidic circuit 120 comprises a plurality ofidentical microfluidic sequestration pens.

In some embodiments, the microfluidic circuit 120 comprises a pluralityof microfluidic sequestration pens, wherein two or more of thesequestration pens comprise differing structures and/or features whichprovide differing benefits in producing embryos. One non-limitingexample may include maintaining ova in one type of pen while maintainingsperm in a different type of pen. In another embodiment, at least one ofthe sequestration pens is configured to have electrical contactssuitable for providing electrical activation for an ovum. In yet anotherembodiment, differing types of cells (such as, for example, uterinecells, endometrial cells, PEG (intercalary) cells derived from theuterine tube (e.g., oviduct or Fallopian tube), cumulus cells, or acombination thereof) may be disposed in sequestration pens adjacent to asequestration pen containing an ovum, such that secretions from thesurrounding sequestration pens may diffuse out of each respective penand into the pen containing an ovum, which is not possible withmacroscale in-vitro culturing and fertilization. Microfluidic devicesuseful for producing an embryo may include any of the sequestration pens124, 126, 128, and 130 or variations thereof, and/or may include pensconfigured like those shown in FIGS. 2B, 2C, 2D,2E and 2F, as discussedbelow.

In the embodiment illustrated in FIG. 1A, a single channel 122 and flowpath 106 is shown. However, other embodiments may contain multiplechannels 122, each configured to comprise a flow path 106. Themicrofluidic circuit 120 further comprises an inlet valve or port 107 influid communication with the flow path 106 and fluidic medium 180,whereby fluidic medium 180 can access channel 122 via the inlet port107. In some instances, the flow path 106 comprises a single path. Insome instances, the single path is arranged in a zigzag pattern wherebythe flow path 106 travels across the microfluidic device 100 two or moretimes in alternating directions.

In some instances, microfluidic circuit 120 comprises a plurality ofparallel channels 122 and flow paths 106, wherein the fluidic medium 180within each flow path 106 flows in the same direction. In someinstances, the fluidic medium within each flow path 106 flows in atleast one of a forward or reverse direction. In some instances, aplurality of sequestration pens is configured (e.g., relative to achannel 122) such that the sequestration pens can be loaded with targetmicro-objects in parallel.

In some embodiments, microfluidic circuit 120 further comprises one ormore micro-object traps 132. The traps 132 are generally formed in awall forming the boundary of a channel 122, and may be positionedopposite an opening of one or more of the microfluidic sequestrationpens 124, 126, 128, 130. In some embodiments, the traps 132 areconfigured to receive or capture a single micro-object from the flowpath 106. In some embodiments, the traps 132 are configured to receiveor capture a plurality of micro-objects from the flow path 106. In someinstances, the traps 132 comprise a volume approximately equal to thevolume of a single target micro-object.

The traps 132 may further comprise an opening which is configured toassist the flow of targeted micro-objects into the traps 132. In someinstances, the traps 132 comprise an opening having a height and widththat is approximately equal to the dimensions of a single targetmicro-object, whereby larger micro-objects are prevented from enteringinto the micro-object trap. The traps 132 may further comprise otherfeatures configured to assist in retention of targeted micro-objectswithin the trap 132. In some instances, the trap 132 is aligned with andsituated on the opposite side of a channel 122 relative to the openingof a microfluidic sequestration pen, such that upon tilting themicrofluidic device 100 about an axis parallel to the microfluidicchannel 122, the trapped micro-object exits the trap 132 at a trajectorythat causes the micro-object to fall into the opening of thesequestration pen. In some instances, the trap 132 comprises a sidepassage 134 that is smaller than the target micro-object in order tofacilitate flow through the trap 132 and thereby increase the likelihoodof capturing a micro-object in the trap 132.

In some embodiments, dielectrophoretic (DEP) forces are applied acrossthe fluidic medium 180 (e.g., in the flow path and/or in thesequestration pens) via one or more electrodes (not shown) tomanipulate, transport, separate and sort micro-objects located therein.For example, in some embodiments, DEP forces are applied to one or moreportions of microfluidic circuit 120 in order to transfer a singlemicro-object from the flow path 106 into a desired microfluidicsequestration pen. In some embodiments, DEP forces are used to prevent amicro-object within a sequestration pen (e.g., sequestration pen 124,126, 128, or 130) from being displaced therefrom. Further, in someembodiments, DEP forces are used to selectively remove a micro-objectfrom a sequestration pen that was previously collected in accordancewith the embodiments of the current disclosure. In some embodiments, theDEP forces comprise optoelectronic tweezer (OET) forces.

In other embodiments, optoelectrowetting (OEW) forces are applied to oneor more positions in the support structure 104 (and/or the cover 110) ofthe microfluidic device 100 (e.g., positions helping to define the flowpath and/or the sequestration pens) via one or more electrodes (notshown) to manipulate, transport, separate and sort droplets located inthe microfluidic circuit 120. For example, in some embodiments, OEWforces are applied to one or more positions in the support structure 104(and/or the cover 110) in order to transfer a single droplet from theflow path 106 into a desired microfluidic sequestration pen. In someembodiments, OEW forces are used to prevent a droplet within asequestration pen (e.g., sequestration pen 124, 126, 128, or 130) frombeing displaced therefrom. Further, in some embodiments, OEW forces areused to selectively remove a droplet from a sequestration pen that waspreviously collected in accordance with the embodiments of the currentdisclosure.

In some embodiments, DEP and/or OEW forces are combined with otherforces, such as flow and/or gravitational force, so as to manipulate,transport, separate and sort micro-objects and/or droplets within themicrofluidic circuit 120. For example, the enclosure 102 can be tilted(e.g., by tilting device 190) to position the flow path 106 andmicro-objects located therein above the microfluidic sequestration pens,and the force of gravity can transport the micro-objects and/or dropletsinto the pens. In some embodiments, the DEP and/or OEW forces can beapplied prior to the other forces. In other embodiments, the DEP and/orOEW forces can be applied after the other forces. In still otherinstances, the DEP and/or OEW forces can be applied at the same time asthe other forces or in an alternating manner with the other forces.

FIGS. 1B, 1C, and 2A-2H illustrates various embodiments of microfluidicdevices that can be used in the practice of the embodiments of thepresent disclosure. FIG. 1B depicts an embodiment in which themicrofluidic device 200 is configured as an optically-actuatedelectrokinetic device. A variety of optically-actuated electrokineticdevices are known in the art, including devices having an optoelectronictweezer (OET) configuration and devices having an opto-electrowetting(OEW) configuration. Examples of suitable OET configurations areillustrated in the following U.S. patent documents, each of which isincorporated herein by reference in its entirety: U.S. Pat. No. RE44,711 (Wu et al.) (originally issued as U.S. Pat. No. 7,612,355); andU.S. Pat. No. 7,956,339 (Ohta et al.). Examples of OEW configurationsare illustrated in U.S. Pat. No. 6,958,132 (Chiou et al.) and U.S.Patent Application Publication No. 2012/0024708 (Chiou et al.), both ofwhich are incorporated by reference herein in their entirety. Yetanother example of an optically-actuated electrokinetic device includesa combined OET/OEW configuration, examples of which are shown in U.S.Patent Publication Nos. 20150306598 (Khandros et al.) and 20150306599(Khandros et al.) and their corresponding PCT Publications WO2015/164846and WO2015/164847, all of which are incorporated herein by reference intheir entirety.

Examples of microfluidic devices having pens in which oocytes, ova, orembryos can be placed, cultured, and/or monitored have been described,for example, in US 2014/0116881 (application Ser. No. 14/060,117, filedOct. 22, 2013), US 2015/0151298 (application no. Ser. No. 14/520,568,filed Oct. 22, 2014), and US 2015/0165436 (application no. Ser. No.14/521,447, filed Oct. 22, 2014), each of which is incorporated hereinby reference in its entirety. U.S. application Ser. Nos. 14/520,568 and14/521,447 also describe exemplary methods of analyzing secretions ofcells cultured in a microfluidic device. Each of the foregoingapplications further describes microfluidic devices configured toproduce dielectrophoretic (DEP) forces, such as optoelectronic tweezers(OET) or configured to provide opto-electro wetting (OEW). For example,the optoelectronic tweezers device illustrated in FIG. 2 of US2014/0116881 is an example of a device that can be utilized inembodiments of the present disclosure to select and move an individualbiological micro-object or a group of biological micro-objects.

Microfluidic Device Motive Configurations.

As described above, the control and monitoring equipment of the systemcan comprise a motive module for selecting and moving objects, such asmicro-objects or droplets, in the microfluidic circuit of a microfluidicdevice. The microfluidic device can have a variety of motiveconfigurations, depending upon the type of object being moved and otherconsiderations. For example, a dielectrophoresis (DEP) configuration canbe utilized to select and move micro-objects in the microfluidiccircuit. Thus, the support structure 104 and/or cover 110 of themicrofluidic device 100 can comprise a DEP configuration for selectivelyinducing DEP forces on micro-objects in a fluidic medium 180 in themicrofluidic circuit 120 and thereby select, capture, and/or moveindividual micro-objects or groups of micro-objects. Alternatively, thesupport structure 104 and/or cover 110 of the microfluidic device 100can comprise an electrowetting (EW) configuration for selectivelyinducing EW forces on droplets in a fluidic medium 180 in themicrofluidic circuit 120 and thereby select, capture, and/or moveindividual droplets or groups of droplets.

One example of a microfluidic device 200 comprising a DEP configurationis illustrated in FIGS. 1B and 1C. While for purposes of simplicityFIGS. 1B and 1C show a side cross-sectional view and a topcross-sectional view, respectively, of a portion of an enclosure 102 ofthe microfluidic device 200 having an open region/chamber 202, it shouldbe understood that the region/chamber 202 may be part of a fluidiccircuit element having a more detailed structure, such as a growthchamber, a sequestration pen, a flow region, or a flow channel.Furthermore, the microfluidic device 200 may include other fluidiccircuit elements. For example, the microfluidic device 200 can include aplurality of growth chambers or sequestration pens and/or one or moreflow regions or flow channels, such as those described herein withrespect to microfluidic device 100. A DEP configuration may beincorporated into any such fluidic circuit elements of the microfluidicdevice 200, or select portions thereof. It should be further appreciatedthat any of the above or below described microfluidic device componentsand system components may be incorporated in and/or used in combinationwith the microfluidic device 200. For example, system 150 includingcontrol and monitoring equipment 152, described above, may be used withmicrofluidic device 200, including one or more of the media module 160,motive module 162, imaging module 164, tilting module 166, and othermodules 168.

As seen in FIG. 1B, the microfluidic device 200 includes a supportstructure 104 having a bottom electrode 204 and an electrode activationsubstrate 206 overlying the bottom electrode 204, and a cover 110 havinga top electrode 210, with the top electrode 210 spaced apart from thebottom electrode 204. The top electrode 210 and the electrode activationsubstrate 206 define opposing surfaces of the region/chamber 202. Amedium 180 contained in the region/chamber 202 thus provides a resistiveconnection between the top electrode 210 and the electrode activationsubstrate 206. A power source 212 configured to be connected to thebottom electrode 204 and the top electrode 210 and create a biasingvoltage between the electrodes, as required for the generation of DEPforces in the region/chamber 202, is also shown. The power source 212can be, for example, an alternating current (AC) power source.

In certain embodiments, the microfluidic device 200 illustrated in FIGS.1B and 1C can have an optically-actuated DEP configuration. Accordingly,changing patterns of light 218 from the light source 216, which may becontrolled by the motive module 162, can selectively activate anddeactivate changing patterns of DEP electrodes at regions 214 of theinner surface 208 of the electrode activation substrate 206.(Hereinafter the regions 214 of a microfluidic device having a DEPconfiguration are referred to as “DEP electrode regions.”) Asillustrated in FIG. 1C, a light pattern 218 directed onto the innersurface 208 of the electrode activation substrate 206 can illuminateselect DEP electrode regions 214 a (shown in white) in a pattern, suchas a square. The non-illuminated DEP electrode regions 214(cross-hatched) are hereinafter referred to as “dark” DEP electroderegions 214. The relative electrical impedance through the DEP electrodeactivation substrate 206 (i.e., from the bottom electrode 204 up to theinner surface 208 of the electrode activation substrate 206 whichinterfaces with the medium 180 in the flow region 106) is greater thanthe relative electrical impedance through the medium 180 in theregion/chamber 202 (i.e., from the inner surface 208 of the electrodeactivation substrate 206 to the top electrode 210 of the cover 110) ateach dark DEP electrode region 214. An illuminated DEP electrode region214 a, however, exhibits a reduced relative impedance through theelectrode activation substrate 206 that is less than the relativeimpedance through the medium 180 in the region/chamber 202 at eachilluminated DEP electrode region 214 a.

With the power source 212 activated, the foregoing DEP configurationcreates an electric field gradient in the fluidic medium 180 betweenilluminated DEP electrode regions 214 a and adjacent dark DEP electroderegions 214, which in turn creates local DEP forces that attract orrepel nearby micro-objects (not shown) in the fluidic medium 180. DEPelectrodes that attract or repel micro-objects in the fluidic medium 180can thus be selectively activated and deactivated at many different suchDEP electrode regions 214 at the inner surface 208 of the region/chamber202 by changing light patterns 218 projected from a light source 216into the microfluidic device 200. Whether the DEP forces attract orrepel nearby micro-objects can depend on such parameters as thefrequency of the power source 212 and the dielectric properties of themedium 180 and/or micro-objects (not shown).

The square pattern 220 of illuminated DEP electrode regions 214 aillustrated in FIG. 1C is an example only. Any pattern of the DEPelectrode regions 214 can be illuminated (and thereby activated) by thepattern of light 218 projected into the microfluidic device 200, and thepattern of illuminated/activated DEP electrode regions 214 can berepeatedly changed by changing or moving the light pattern 218.

In some embodiments, the electrode activation substrate 206 can compriseor consist of a photoconductive material. In such embodiments, the innersurface 208 of the electrode activation substrate 206 can befeatureless. For example, the electrode activation substrate 206 cancomprise or consist of a layer of hydrogenated amorphous silicon(a-Si:H). The a-Si:H can comprise, for example, about 8% to 40% hydrogen(calculated as 100*the number of hydrogen atoms/the total number ofhydrogen and silicon atoms). The layer of a-Si:H can have a thickness ofabout 500 nm to about 2.0 μm. In such embodiments, the DEP electroderegions 214 can be created anywhere and in any pattern on the innersurface 208 of the electrode activation substrate 206, in accordancewith the light pattern 218. The number and pattern of the DEP electroderegions 214 thus need not be fixed, but can correspond to the lightpattern 218. Examples of microfluidic devices having a DEP configurationcomprising a photoconductive layer such as discussed above have beendescribed, for example, in U.S. Pat. No. RE 44,711 (Wu et al.)(originally issued as U.S. Pat. No. 7,612,355), the entire contents ofwhich are incorporated herein by reference.

In other embodiments, the electrode activation substrate 206 cancomprise a substrate comprising a plurality of doped layers,electrically insulating layers (or regions), and electrically conductivelayers that form semiconductor integrated circuits, such as is known insemiconductor fields. For example, the electrode activation substrate206 can comprise a plurality of phototransistors, including, forexample, lateral bipolar phototransistors, each phototransistorcorresponding to a DEP electrode region 214. Alternatively, theelectrode activation substrate 206 can comprise electrodes (e.g.,conductive metal electrodes) controlled by phototransistor switches,with each such electrode corresponding to a DEP electrode region 214.The electrode activation substrate 206 can include a pattern of suchphototransistors or phototransistor-controlled electrodes. The pattern,for example, can be an array of substantially square phototransistors orphototransistor-controlled electrodes arranged in rows and columns, suchas shown in FIG. 2B. Alternatively, the pattern can be an array ofsubstantially hexagonal phototransistors or phototransistor-controlledelectrodes that form a hexagonal lattice. Regardless of the pattern,electric circuit elements can form electrical connections between theDEP electrode regions 214 at the inner surface 208 of the electrodeactivation substrate 206 and the bottom electrode 210, and thoseelectrical connections (i.e., phototransistors or electrodes) can beselectively activated and deactivated by the light pattern 218. When notactivated, each electrical connection can have high impedance such thatthe relative impedance through the electrode activation substrate 206(i.e., from the bottom electrode 204 to the inner surface 208 of theelectrode activation substrate 206 which interfaces with the medium 180in the region/chamber 202) is greater than the relative impedancethrough the medium 180 (i.e., from the inner surface 208 of theelectrode activation substrate 206 to the top electrode 210 of the cover110) at the corresponding DEP electrode region 214. When activated bylight in the light pattern 218, however, the relative impedance throughthe electrode activation substrate 206 is less than the relativeimpedance through the medium 180 at each illuminated DEP electroderegion 214, thereby activating the DEP electrode at the correspondingDEP electrode region 214 as discussed above. DEP electrodes that attractor repel micro-objects (not shown) in the medium 180 can thus beselectively activated and deactivated at many different DEP electroderegions 214 at the inner surface 208 of the electrode activationsubstrate 206 in the region/chamber 202 in a manner determined by thelight pattern 218.

Examples of microfluidic devices having electrode activation substratesthat comprise phototransistors have been described, for example, in U.S.Pat. No. 7,956,339 (Ohta et al.) (see, e.g., device 300 illustrated inFIGS. 21 and 22, and descriptions thereof), the entire contents of whichare incorporated herein by reference. Examples of microfluidic deviceshaving electrode activation substrates that comprise electrodescontrolled by phototransistor switches have been described, for example,in U.S. Patent Publication No. 2014/0124370 (Short et al.) (see, e.g.,devices 200, 400, 500, 600, and 900 illustrated throughout the drawings,and descriptions thereof), the entire contents of which are incorporatedherein by reference.

In some embodiments of a DEP configured microfluidic device, the topelectrode 210 is part of a first wall (or cover 110) of the enclosure102, and the electrode activation substrate 206 and bottom electrode 204are part of a second wall (or support structure 104) of the enclosure102. The region/chamber 202 can be between the first wall and the secondwall. In other embodiments, the electrode 210 is part of the second wall(or support structure 104) and one or both of the electrode activationsubstrate 206 and/or the electrode 210 are part of the first wall (orcover 110). Moreover, the light source 216 can alternatively be used toilluminate the enclosure 102 from below.

With the microfluidic device 200 of FIGS. 1B-1C having a DEPconfiguration, the motive module 162 can select a micro-object (notshown) in the medium 180 in the region/chamber 202 by projecting a lightpattern 218 into the microfluidic device 200 to activate a first set ofone or more DEP electrodes at DEP electrode regions 214 a of the innersurface 208 of the electrode activation substrate 206 in a pattern(e.g., square pattern 220) that surrounds and captures the micro-object.The motive module 162 can then move the in situ-generated capturedmicro-object by moving the light pattern 218 relative to themicrofluidic device 200 to activate a second set of one or more DEPelectrodes at DEP electrode regions 214. Alternatively, the microfluidicdevice 200 can be moved relative to the light pattern 218.

In other embodiments, the microfluidic device 200 can have a DEPconfiguration that does not rely upon light activation of DEP electrodesat the inner surface 208 of the electrode activation substrate 206. Forexample, the electrode activation substrate 206 can comprise selectivelyaddressable and energizable electrodes positioned opposite to a surfaceincluding at least one electrode (e.g., cover 110). Switches (e.g.,transistor switches in a semiconductor substrate) may be selectivelyopened and closed to activate or inactivate DEP electrodes at DEPelectrode regions 214, thereby creating a net DEP force on amicro-object (not shown) in region/chamber 202 in the vicinity of theactivated DEP electrodes. Depending on such characteristics as thefrequency of the power source 212 and the dielectric properties of themedium (not shown) and/or micro-objects in the region/chamber 202, theDEP force can attract or repel a nearby micro-object. By selectivelyactivating and deactivating a set of DEP electrodes (e.g., at a set ofDEP electrodes regions 214 that forms a square pattern 220), one or moremicro-objects in region/chamber 202 can be trapped and moved within theregion/chamber 202. The motive module 162 in FIG. 1A can control suchswitches and thus activate and deactivate individual ones of the DEPelectrodes to select, trap, and move particular micro-objects (notshown) around the region/chamber 202. Microfluidic devices having a DEPconfiguration that includes selectively addressable and energizableelectrodes are known in the art and have been described, for example, inU.S. Pat. No. 6,294,063 (Becker et al.) and U.S. Pat. No. 6,942,776(Medoro), the entire contents of which are incorporated herein byreference.

As yet another example, the microfluidic device 200 can have anelectrowetting (EW) configuration, which can be in place of the DEPconfiguration or can be located in a portion of the microfluidic device200 that is separate from the portion which has the DEP configuration.The EW configuration can be an opto-electrowetting configuration or anelectrowetting on dielectric (EWOD) configuration, both of which areknown in the art. In some EW configurations, the support structure 104has an electrode activation substrate 206 sandwiched between adielectric layer (not shown) and the bottom electrode 204. Thedielectric layer can comprise a hydrophobic material and/or can becoated with a hydrophobic material, as described below. For microfluidicdevices 200 that have an EW configuration, the inner surface 208 of thesupport structure 104 is the inner surface of the dielectric layer orits hydrophobic coating.

The dielectric layer (not shown) can comprise one or more oxide layers,and can have a thickness of about 50 nm to about 250 nm (e.g., about 125nm to about 175 nm). In certain embodiments, the dielectric layer maycomprise a layer of oxide, such as a metal oxide (e.g., aluminum oxideor hafnium oxide). In certain embodiments, the dielectric layer cancomprise a dielectric material other than a metal oxide, such as siliconoxide or a nitride. Regardless of the exact composition and thickness,the dielectric layer can have an impedance of about 10 kOhms to about 50kOhms.

In some embodiments, the surface of the dielectric layer that facesinward toward region/chamber 202 is coated with a hydrophobic material.The hydrophobic material can comprise, for example, fluorinated carbonmolecules. Examples of fluorinated carbon molecules includeperfluoro-polymers such as polytetrafluoroethylene (e.g., TEFLON®) orpoly(2,3-difluoromethylenyl-perfluorotetrahydrofuran) (e.g., CYTOP™).Molecules that make up the hydrophobic material can be covalently bondedto the surface of the dielectric layer. For example, molecules of thehydrophobic material can be covalently bound to the surface of thedielectric layer by means of a linker such as a siloxane group, aphosphonic acid group, or a thiol group. Thus, in some embodiments, thehydrophobic material can comprise alkyl-terminated siloxane,alkyl-termination phosphonic acid, or alkyl-terminated thiol. The alkylgroup can be long-chain hydrocarbons (e.g., having a chain of at least10 carbons, or at least 16, 18, 20, 22, or more carbons). Alternatively,fluorinated (or perfluorinated) carbon chains can be used in place ofthe alkyl groups. Thus, for example, the hydrophobic material cancomprise fluoroalkyl-terminated siloxane, fluoroalkyl-terminatedphosphonic acid, or fluoroalkyl-terminated thiol. In some embodiments,the hydrophobic coating has a thickness of about 10 nm to about 50 nm.In other embodiments, the hydrophobic coating has a thickness of lessthan 10 nm (e.g., less than 5 nm, or about 1.5 to 3.0 nm).

In some embodiments, the cover 110 of a microfluidic device 200 havingan electrowetting configuration is coated with a hydrophobic material(not shown) as well. The hydrophobic material can be the samehydrophobic material used to coat the dielectric layer of the supportstructure 104, and the hydrophobic coating can have a thickness that issubstantially the same as the thickness of the hydrophobic coating onthe dielectric layer of the support structure 104. Moreover, the cover110 can comprise an electrode activation substrate 206 sandwichedbetween a dielectric layer and the top electrode 210, in the manner ofthe support structure 104. The electrode activation substrate 206 andthe dielectric layer of the cover 110 can have the same compositionand/or dimensions as the electrode activation substrate 206 and thedielectric layer of the support structure 104. Thus, the microfluidicdevice 200 can have two electrowetting surfaces.

In some embodiments, the electrode activation substrate 206 can comprisea photoconductive material, such as described above. Accordingly, incertain embodiments, the electrode activation substrate 206 can compriseor consist of a layer of hydrogenated amorphous silicon (a-Si:H). Thea-Si:H can comprise, for example, about 8% to 40% hydrogen (calculatedas 100*the number of hydrogen atoms/the total number of hydrogen andsilicon atoms). The layer of a-Si:H can have a thickness of about 500 nmto about 2.0 μm. Alternatively, the electrode activation substrate 206can comprise electrodes (e.g., conductive metal electrodes) controlledby phototransistor switches, as described above. Microfluidic deviceshaving an opto-electrowetting configuration are known in the art and/orcan be constructed with electrode activation substrates known in theart. For example, U.S. Pat. No. 6,958,132 (Chiou et al.), the entirecontents of which are incorporated herein by reference, disclosesopto-electrowetting configurations having a photoconductive materialsuch as a-Si:H, while U.S. Patent Publication No. 2014/0124370 (Short etal.), referenced above, discloses electrode activation substrates havingelectrodes controlled by phototransistor switches.

The microfluidic device 200 thus can have an opto-electrowettingconfiguration, and light patterns 218 can be used to activatephotoconductive EW regions or photoresponsive EW electrodes in theelectrode activation substrate 206. Such activated EW regions or EWelectrodes of the electrode activation substrate 206 can generate anelectrowetting force at the inner surface 208 of the support structure104 (i.e., the inner surface of the overlaying dielectric layer or itshydrophobic coating). By changing the light patterns 218 (or movingmicrofluidic device 200 relative to the light source 216) incident onthe electrode activation substrate 206, droplets (e.g., containing anaqueous medium, solution, or solvent) contacting the inner surface 208of the support structure 104 can be moved through an immiscible fluid(e.g., an oil medium) present in the region/chamber 202.

In other embodiments, microfluidic devices 200 can have an EWODconfiguration, and the electrode activation substrate 206 can compriseselectively addressable and energizable electrodes that do not rely uponlight for activation. The electrode activation substrate 206 thus caninclude a pattern of such electrowetting (EW) electrodes. The pattern,for example, can be an array of substantially square EW electrodesarranged in rows and columns, such as shown in FIG. 2B. Alternatively,the pattern can be an array of substantially hexagonal EW electrodesthat form a hexagonal lattice. Regardless of the pattern, the EWelectrodes can be selectively activated (or deactivated) by electricalswitches (e.g., transistor switches in a semiconductor substrate). Byselectively activating and deactivating EW electrodes in the electrodeactivation substrate 206, droplets (not shown) contacting the innersurface 208 of the overlaying dielectric layer or its hydrophobiccoating can be moved within the region/chamber 202. The motive module162 in FIG. 1A can control such switches and thus activate anddeactivate individual EW electrodes to select and move particulardroplets around region/chamber 202. Microfluidic devices having a EWODconfiguration with selectively addressable and energizable electrodesare known in the art and have been described, for example, in U.S. Pat.No. 8,685,344 (Sundarsan et al.), the entire contents of which areincorporated herein by reference.

Regardless of the configuration of the microfluidic device 200, a powersource 212 can be used to provide a potential (e.g., an AC voltagepotential) that powers the electrical circuits of the microfluidicdevice 200. The power source 212 can be the same as, or a component of,the power source 192 referenced in FIG. 1. Power source 212 can beconfigured to provide an AC voltage and/or current to the top electrode210 and the bottom electrode 204. For an AC voltage, the power source212 can provide a frequency range and an average or peak power (e.g.,voltage or current) range sufficient to generate net DEP forces (orelectrowetting forces) strong enough to trap and move individualmicro-objects (not shown) in the region/chamber 202, as discussed above,and/or to change the wetting properties of the inner surface 208 of thesupport structure 104 (i.e., the dielectric layer and/or the hydrophobiccoating on the dielectric layer) in the region/chamber 202, as alsodiscussed above. Such frequency ranges and average or peak power rangesare known in the art. See, e.g., U.S. Pat. No. 6,958,132 (Chiou et al.),U.S. Pat. No. RE44,711 (Wu et al.) (originally issued as U.S. Pat. No.7,612,355), and US Patent Application Publication Nos. US2014/0124370(Short et al.), US2015/0306598 (Khandros et al.), and US2015/0306599(Khandros et al.).

Sequestration Pens.

Non-limiting examples of generic sequestration pens 224, 226, and 228are shown within the microfluidic device 230 depicted in FIGS. 2A-2C.Each sequestration pen 224, 226, and 228 can comprise an isolationstructure 232 defining an isolation region 240 and a connection region236 fluidically connecting the isolation region 240 to a channel 122.The connection region 236 can comprise a proximal opening 234 to themicrofluidic channel 122 and a distal opening 238 to the isolationregion 240. The connection region 236 can be configured so that themaximum penetration depth of a flow of a fluidic medium (not shown)flowing from the microfluidic channel 122 into the sequestration pen224, 226, 228 does not extend into the isolation region 240. Thus, dueto the connection region 236, a micro-object (not shown) or othermaterial (not shown) disposed in an isolation region 240 of asequestration pen 224, 226, 228 can thus be isolated from, and notsubstantially affected by, a flow of medium 180 in the microfluidicchannel 122.

The sequestration pens 224, 226, and 228 of FIGS. 2A-2C each have asingle opening which opens directly to the microfluidic channel 122. Theopening of the sequestration pen opens laterally from the microfluidicchannel 122. The electrode activation substrate 206 underlays both themicrofluidic channel 122 and the sequestration pens 224, 226, and 228.The upper surface of the electrode activation substrate 206 within theenclosure of a sequestration pen, forming the floor of the sequestrationpen, is disposed at the same level or substantially the same level ofthe upper surface the of electrode activation substrate 206 within themicrofluidic channel 122 (or flow region if a channel is not present),forming the floor of the flow channel (or flow region, respectively) ofthe microfluidic device. The electrode activation substrate 206 may befeatureless or may have an irregular or patterned surface that variesfrom its highest elevation to its lowest depression by less than about 3microns, 2.5 microns, 2 microns, 1.5 microns, 1 micron, 0.9 microns, 0.5microns, 0.4 microns, 0.2 microns, 0.1 microns or less. The variation ofelevation in the upper surface of the substrate across both themicrofluidic channel 122 (or flow region) and sequestration pens may beless than about 3%, 2%, 1%. 0.9%, 0.8%, 0.5%, 0.3% or 0.1% of the heightof the walls of the sequestration pen or walls of the microfluidicdevice. While described in detail for the microfluidic device 200, thisalso applies to any of the microfluidic devices 100, 230, 250, 280, 290,320, 400, 450, 500, 700 described herein.

The microfluidic channel 122 can thus be an example of a swept region,and the isolation regions 240 of the sequestration pens 224, 226, 228can be examples of unswept regions. As noted, the microfluidic channel122 and sequestration pens 224, 226, 228 can be configured to containone or more fluidic media 180. In the example shown in FIGS. 2A-2B, theports 222 are connected to the microfluidic channel 122 and allow afluidic medium 180 to be introduced into or removed from themicrofluidic device 230. Prior to introduction of the fluidic medium180, the microfluidic device may be primed with a gas such as carbondioxide gas. Once the microfluidic device 230 contains the fluidicmedium 180, the flow 242 of fluidic medium 180 in the microfluidicchannel 122 can be selectively generated and stopped. For example, asshown, the ports 222 can be disposed at different locations (e.g.,opposite ends) of the microfluidic channel 122, and a flow 242 of mediumcan be created from one port 222 functioning as an inlet to another port222 functioning as an outlet.

FIG. 2C illustrates a detailed view of an example of a sequestration pen224 according to the present disclosure. Examples of micro-objects 246are also shown.

As is known, a flow 242 of fluidic medium 180 in a microfluidic channel122 past a proximal opening 234 of sequestration pen 224 can cause asecondary flow 244 of the medium 180 into and/or out of thesequestration pen 224. To isolate micro-objects 246 in the isolationregion 240 of a sequestration pen 224 from the secondary flow 244, thelength L_(con) of the connection region 236 of the sequestration pen 224(i.e., from the proximal opening 234 to the distal opening 238) shouldbe greater than the penetration depth D_(p) of the secondary flow 244into the connection region 236. The penetration depth D_(p) of thesecondary flow 244 depends upon the velocity of the fluidic medium 180flowing in the microfluidic channel 122 and various parameters relatingto the configuration of the microfluidic channel 122 and the proximalopening 234 of the connection region 236 to the microfluidic channel122. For a given microfluidic device, the configurations of themicrofluidic channel 122 and the opening 234 will be fixed, whereas therate of flow 242 of fluidic medium 180 in the microfluidic channel 122will be variable. Accordingly, for each sequestration pen 224, a maximalvelocity V_(max) for the flow 242 of fluidic medium 180 in channel 122can be identified that ensures that the penetration depth D_(p) of thesecondary flow 244 does not exceed the length L_(con) of the connectionregion 236. As long as the rate of the flow 242 of fluidic medium 180 inthe microfluidic channel 122 does not exceed the maximum velocityV_(max), the resulting secondary flow 244 can be limited to themicrofluidic channel 122 and the connection region 236 and kept out ofthe isolation region 240. The flow 242 of medium 180 in the microfluidicchannel 122 will thus not draw micro-objects 246 out of the isolationregion 240. Rather, micro-objects 246 located in the isolation region240 will stay in the isolation region 240 regardless of the flow 242 offluidic medium 180 in the microfluidic channel 122.

Moreover, as long as the rate of flow 242 of medium 180 in themicrofluidic channel 122 does not exceed V_(max), the flow 242 offluidic medium 180 in the microfluidic channel 122 will not movemiscellaneous particles (e.g., microparticles and/or nanoparticles) fromthe microfluidic channel 122 into the isolation region 240 of asequestration pen 224. Having the length L_(con) of the connectionregion 236 be greater than the maximum penetration depth D_(p) of thesecondary flow 244 can thus prevent contamination of one sequestrationpen 224 with miscellaneous particles from the microfluidic channel 122or another sequestration pen (e.g., sequestration pens 226, 228 in FIG.2D).

Because the microfluidic channel 122 and the connection regions 236 ofthe sequestration pens 224, 226, 228 can be affected by the flow 242 ofmedium 180 in the microfluidic channel 122, the microfluidic channel 122and connection regions 236 can be deemed swept (or flow) regions of themicrofluidic device 230. The isolation regions 240 of the sequestrationpens 224, 226, 228, on the other hand, can be deemed unswept (ornon-flow) regions. For example, components (not shown) in a firstfluidic medium 180 in the microfluidic channel 122 can mix with a secondfluidic medium 248 in the isolation region 240 substantially only bydiffusion of components of the first medium 180 from the microfluidicchannel 122 through the connection region 236 and into the secondfluidic medium 248 in the isolation region 240. Similarly, components(not shown) of the second medium 248 in the isolation region 240 can mixwith the first medium 180 in the microfluidic channel 122 substantiallyonly by diffusion of components of the second medium 248 from theisolation region 240 through the connection region 236 and into thefirst medium 180 in the microfluidic channel 122. In some embodiments,the extent of fluidic medium exchange between the isolation region of asequestration pen and the flow region by diffusion is greater than about90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or greater than about 99% offluidic exchange. The first medium 180 can be the same medium or adifferent medium than the second medium 248. Moreover, the first medium180 and the second medium 248 can start out being the same, then becomedifferent (e.g., through conditioning of the second medium 248 by one ormore cells in the isolation region 240, or by changing the medium 180flowing through the microfluidic channel 122).

The maximum penetration depth D_(p) of the secondary flow 244 caused bythe flow 242 of fluidic medium 180 in the microfluidic channel 122 candepend on a number of parameters, as mentioned above. Examples of suchparameters include: the shape of the microfluidic channel 122 (e.g., themicrofluidic channel can direct medium into the connection region 236,divert medium away from the connection region 236, or direct medium in adirection substantially perpendicular to the proximal opening 234 of theconnection region 236 to the microfluidic channel 122); a width W_(ch)(or cross-sectional area) of the microfluidic channel 122 at theproximal opening 234; and a width W_(con) (or cross-sectional area) ofthe connection region 236 at the proximal opening 234; the velocity V ofthe flow 242 of fluidic medium 180 in the microfluidic channel 122; theviscosity of the first medium 180 and/or the second medium 248, or thelike.

In some embodiments, the dimensions of the microfluidic channel 122 andsequestration pens 224, 226, 228 can be oriented as follows with respectto the vector of the flow 242 of fluidic medium 180 in the microfluidicchannel 122: the microfluidic channel width W_(ch) (or cross-sectionalarea of the microfluidic channel 122) can be substantially perpendicularto the flow 242 of medium 180; the width W_(con) (or cross-sectionalarea) of the connection region 236 at opening 234 can be substantiallyparallel to the flow 242 of medium 180 in the microfluidic channel 122;and/or the length Lon of the connection region can be substantiallyperpendicular to the flow 242 of medium 180 in the microfluidic channel122. The foregoing are examples only, and the relative position of themicrofluidic channel 122 and sequestration pens 224, 226, 228 can be inother orientations with respect to each other.

As illustrated in FIG. 2C, the width W_(con) of the connection region236 can be uniform from the proximal opening 234 to the distal opening238. The width W_(con) of the connection region 236 at the distalopening 238 can thus be in any of the ranges identified herein for thewidth W_(con) of the connection region 236 at the proximal opening 234.Alternatively, the width W_(con) of the connection region 236 at thedistal opening 238 can be larger than the width W_(con) of theconnection region 236 at the proximal opening 234.

As illustrated in FIG. 2C, the width of the isolation region 240 at thedistal opening 238 can be substantially the same as the width W_(con) ofthe connection region 236 at the proximal opening 234. The width of theisolation region 240 at the distal opening 238 can thus be in any of theranges identified herein for the width W_(con) of the connection region236 at the proximal opening 234. Alternatively, the width of theisolation region 240 at the distal opening 238 can be larger or smallerthan the width W_(con) of the connection region 236 at the proximalopening 234. Moreover, the distal opening 238 may be smaller than theproximal opening 234 and the width W_(con) of the connection region 236may be narrowed between the proximal opening 234 and distal opening 238.For example, the connection region 236 may be narrowed between theproximal opening and the distal opening, using a variety of differentgeometries (e.g. chamfering the connection region, beveling theconnection region). Further, any part or subpart of the connectionregion 236 may be narrowed (e.g. a portion of the connection regionadjacent to the proximal opening 234).

FIGS. 2D-2F depict another exemplary embodiment of a microfluidic device250 containing a microfluidic circuit 262 and flow channels 264, whichare variations of the respective microfluidic device 100, circuit 132and channel 134 of FIG. 1A. The microfluidic device 250 also has aplurality of sequestration pens 266 that are additional variations ofthe above-described sequestration pens 124, 126, 128, 130, 224, 226 or228. In particular, it should be appreciated that the sequestration pens266 of device 250 shown in FIGS. 2D-2F can replace any of theabove-described sequestration pens 124, 126, 128, 130, 224, 226 or 228in devices 100, 200, 230, 280, 290, 320, 400, 450, 500, 700. Likewise,the microfluidic device 250 is another variant of the microfluidicdevice 100, and may also have the same or a different DEP configurationas the above-described microfluidic device 100, 200, 230, 280, 290, 320,400, 450, 500, 700 as well as any of the other microfluidic systemcomponents described herein.

The microfluidic device 250 of FIGS. 2D-2F comprises a support structure(not visible in FIGS. 2D-2F, but can be the same or generally similar tothe support structure 104 of device 100 depicted in FIG. 1A), amicrofluidic circuit structure 256, and a cover (not visible in FIGS.2D-2F, but can be the same or generally similar to the cover 122 ofdevice 100 depicted in FIG. 1A). The microfluidic circuit structure 256includes a frame 252 and microfluidic circuit material 260, which can bethe same as or generally similar to the frame 114 and microfluidiccircuit material 116 of device 100 shown in FIG. 1A. As shown in FIG.2D, the microfluidic circuit 262 defined by the microfluidic circuitmaterial 260 can comprise multiple channels 264 (two are shown but therecan be more) to which multiple sequestration pens 266 are fluidicallyconnected.

Each sequestration pen 266 can comprise an isolation structure 272, anisolation region 270 within the isolation structure 272, and aconnection region 268. From a proximal opening 274 at the microfluidicchannel 264 to a distal opening 276 at the isolation structure 272, theconnection region 268 fluidically connects the microfluidic channel 264to the isolation region 270. Generally, in accordance with the abovediscussion of FIGS. 2B and 2C, a flow 278 of a first fluidic medium 254in a channel 264 can create secondary flows 282 of the first medium 254from the microfluidic channel 264 into and/or out of the respectiveconnection regions 268 of the sequestration pens 266.

As illustrated in FIG. 2E, the connection region 268 of eachsequestration pen 266 generally includes the area extending between theproximal opening 274 to a channel 264 and the distal opening 276 to anisolation structure 272. The length L_(con) of the connection region 268can be greater than the maximum penetration depth D_(p) of secondaryflow 282, in which case the secondary flow 282 will extend into theconnection region 268 without being redirected toward the isolationregion 270 (as shown in FIG. 2D). Alternatively, at illustrated in FIG.2F, the connection region 268 can have a length L_(con) that is lessthan the maximum penetration depth D_(p), in which case the secondaryflow 282 will extend through the connection region 268 and be redirectedtoward the isolation region 270. In this latter situation, the sum oflengths L_(c1) and L_(c2) of connection region 268 is greater than themaximum penetration depth D_(p), so that secondary flow 282 will notextend into isolation region 270. Whether length L_(con) of connectionregion 268 is greater than the penetration depth D_(p), or the sum oflengths L_(c1) and L_(c2) of connection region 268 is greater than thepenetration depth D_(p), a flow 278 of a first medium 254 in channel 264that does not exceed a maximum velocity V_(max) will produce a secondaryflow having a penetration depth D_(p), and micro-objects (not shown butcan be the same or generally similar to the micro-objects 246 shown inFIG. 2C) in the isolation region 270 of a sequestration pen 266 will notbe drawn out of the isolation region 270 by a flow 278 of first medium254 in channel 264. Nor will the flow 278 in channel 264 drawmiscellaneous materials (not shown) from channel 264 into the isolationregion 270 of a sequestration pen 266. As such, diffusion is the onlymechanism by which components in a first medium 254 in the microfluidicchannel 264 can move from the microfluidic channel 264 into a secondmedium 258 in an isolation region 270 of a sequestration pen 266.Likewise, diffusion is the only mechanism by which components in asecond medium 258 in an isolation region 270 of a sequestration pen 266can move from the isolation region 270 to a first medium 254 in themicrofluidic channel 264. The first medium 254 can be the same medium asthe second medium 258, or the first medium 254 can be a different mediumthan the second medium 258. Alternatively, the first medium 254 and thesecond medium 258 can start out being the same, then become different,e.g., through conditioning of the second medium by one or more cells inthe isolation region 270, or by changing the medium flowing through themicrofluidic channel 264.

As illustrated in FIG. 2E, the width W_(ch) of the microfluidic channels264 (i.e., taken transverse to the direction of a fluid medium flowthrough the microfluidic channel indicated by arrows 278 in FIG. 2D) inthe microfluidic channel 264 can be substantially perpendicular to awidth W_(con1) of the proximal opening 274 and thus substantiallyparallel to a width W_(con2) of the distal opening 276. The widthW_(con1) of the proximal opening 274 and the width W_(con2) of thedistal opening 276, however, need not be substantially perpendicular toeach other. For example, an angle between an axis (not shown) on whichthe width W_(con1) of the proximal opening 274 is oriented and anotheraxis on which the width W_(con2) of the distal opening 276 is orientedcan be other than perpendicular and thus other than 90°. Examples ofalternatively oriented angles include angles in any of the followingranges: from about 30° to about 90°, from about 45° to about 90°, fromabout 60° to about 90°, or the like.

In various embodiments of sequestration pens (e.g. 124, 126, 128, 130,224, 226, 228, or 266), the isolation region (e.g. 240 or 270) isconfigured to contain a plurality of micro-objects. In otherembodiments, the isolation region can be configured to contain only one,two, three, four, five, or a similar relatively small number ofmicro-objects. Accordingly, the volume of an isolation region can be,for example, at least 1×10⁶, 2×10⁶, 4×10⁶, 6×10⁶ cubic microns, or more.

In various embodiments of sequestration pens, the width W_(ch) of themicrofluidic channel (e.g., 122) at a proximal opening (e.g. 234) can bewithin any of the following ranges: about 50-1000 microns, 50-500microns, 50-400 microns, 50-300 microns, 50-250 microns, 50-200 microns,50-150 microns, 50-100 microns, 70-500 microns, 70-400 microns, 70-300microns, 70-250 microns, 70-200 microns, 70-150 microns, 90-400 microns,90-300 microns, 90-250 microns, 90-200 microns, 90-150 microns, 100-300microns, 100-250 microns, 100-200 microns, 100-150 microns, and 100-120microns. In some other embodiments, the width W_(ch) of the microfluidicchannel (e.g., 122) at a proximal opening (e.g. 234) can be in a rangeof about 200-800 microns, 200-700 microns, or 200-600 microns. Theforegoing are examples only, and the width W_(ch) of the microfluidicchannel 122 can be in other ranges (e.g., a range defined by any of theendpoints listed above). Moreover, the W_(ch) of the microfluidicchannel 122 can be selected to be in any of these ranges in regions ofthe microfluidic channel other than at a proximal opening of asequestration pen.

In some embodiments, a sequestration pen has a height of about 30 toabout 200 microns, or about 50 to about 150 microns. In someembodiments, the sequestration pen has a cross-sectional area of about1×10⁴-3×10⁶ square microns, 2×10⁴-2×10⁶ square microns, 4×10⁴-1×10⁶square microns, 2×10⁴-5×10⁵ square microns, 2×10⁴-1×10⁵ square micronsor about 2×10⁵-2×10⁶ square microns.

In various embodiments of sequestration pens, the height H_(ch) of themicrofluidic channel (e.g., 122) at a proximal opening (e.g., 234) canbe within any of the following ranges: 20-100 microns, 20-90 microns,20-80 microns, 20-70 microns, 20-60 microns, 20-50 microns, 30-100microns, 30-90 microns, 30-80 microns, 30-70 microns, 30-60 microns,30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-70microns, 40-60 microns, or 40-50 microns. The foregoing are examplesonly, and the height H_(ch) of the microfluidic channel (e.g., 122) canbe in other ranges (e.g., a range defined by any of the endpoints listedabove). The height H_(ch) of the microfluidic channel 122 can beselected to be in any of these ranges in regions of the microfluidicchannel other than at a proximal opening of an sequestration pen.

In various embodiments of sequestration pens a cross-sectional area ofthe microfluidic channel (e.g., 122) at a proximal opening (e.g., 234)can be within any of the following ranges: 500-50,000 square microns,500-40,000 square microns, 500-30,000 square microns, 500-25,000 squaremicrons, 500-20,000 square microns, 500-15,000 square microns,500-10,000 square microns, 500-7,500 square microns, 500-5,000 squaremicrons, 1,000-25,000 square microns, 1,000-20,000 square microns,1,000-15,000 square microns, 1,000-10,000 square microns, 1,000-7,500square microns, 1,000-5,000 square microns, 2,000-20,000 square microns,2,000-15,000 square microns, 2,000-10,000 square microns, 2,000-7,500square microns, 2,000-6,000 square microns, 3,000-20,000 square microns,3,000-15,000 square microns, 3,000-10,000 square microns, 3,000-7,500square microns, or 3,000 to 6,000 square microns. The foregoing areexamples only, and the cross-sectional area of the microfluidic channel(e.g., 122) at a proximal opening (e.g., 234) can be in other ranges(e.g., a range defined by any of the endpoints listed above).

In various embodiments of sequestration pens, the length L_(con) of theconnection region (e.g., 236) can be in any of the following ranges:about 1-600 microns, 5-550 microns, 10-500 microns, 15-400 microns,20-300 microns, 20-500 microns, 40-400 microns, 60-300 microns, 80-200microns, or about 100-150 microns. The foregoing are examples only, andlength L_(con) of a connection region (e.g., 236) can be in a differentrange than the foregoing examples (e.g., a range defined by any of theendpoints listed above).

In various embodiments of sequestration pens the width W_(con) of aconnection region (e.g., 236) at a proximal opening (e.g., 234) can bein any of the following ranges: 20-500 microns, 20-400 microns, 20-300microns, 20-200 microns, 20-150 microns, 20-100 microns, 20-80 microns,20-60 microns, 30-400 microns, 30-300 microns, 30-200 microns, 30-150microns, 30-100 microns, 30-80 microns, 30-60 microns, 40-300 microns,40-200 microns, 40-150 microns, 40-100 microns, 40-80 microns, 40-60microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100 microns,50-80 microns, 60-200 microns, 60-150 microns, 60-100 microns, 60-80microns, 70-150 microns, 70-100 microns, and 80-100 microns. Theforegoing are examples only, and the width W_(con) of a connectionregion (e.g., 236) at a proximal opening (e.g., 234) can be differentthan the foregoing examples (e.g., a range defined by any of theendpoints listed above).

In various embodiments of sequestration pens, the width W_(con) of aconnection region (e.g., 236) at a proximal opening (e.g., 234) can beat least as large as the largest dimension of a micro-object (e.g.,biological cell which may be a T cell, B cell, or an ovum or embryo)that the sequestration pen is intended for. For example, the widthW_(con) of a connection region 236 at a proximal opening 234 of ansequestration pen that an oocyte, ovum, or embryo will be placed intocan be in any of the following ranges: about 100 microns, about 110microns, about 120 microns, about 130 microns, about 140 microns, about150 microns, about 160 microns, about 170 microns, about 180 microns,about 190 microns, about 200 microns, about 225 microns, about 250microns, about 300 microns or about 100-400 microns, about 120-350microns, about 140-200-200 300 microns, or about 140-200 microns. Theforegoing are examples only, and the width W_(con) of a connectionregion (e.g., 236) at a proximal opening (e.g., 234) can be differentthan the foregoing examples (e.g., a range defined by any of theendpoints listed above).

In various embodiments of sequestration pens, the width W_(pr) of aproximal opening of a connection region may be at least as large as thelargest dimension of a micro-object (e.g., a biological micro-objectsuch as a cell) that the sequestration pen is intended for. For example,the width W_(pr) may be about 50 microns, about 60 microns, about 100microns, about 200 microns, about 300 microns or may be in a range ofabout 50-300 microns, about 50-200 microns, about 50-100 microns, about75-150 microns, about 75-100 microns, or about 200-300 microns

In various embodiments of sequestration pens, a ratio of the lengthL_(con) of a connection region (e.g., 236) to a width W_(con) of theconnection region (e.g., 236) at the proximal opening 234 can be greaterthan or equal to any of the following ratios: 0.5, 1.0, 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, or more. Theforegoing are examples only, and the ratio of the length Lon of aconnection region 236 to a width W_(con), of the connection region 236at the proximal opening 234 can be different than the foregoingexamples.

In various embodiments of microfluidic devices 100, 200, 23, 250, 280,290, 320, 400, 450, 500, 700, V_(max) can be set around 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5microliters/sec.

In various embodiments of microfluidic devices having sequestrationpens, the volume of an isolation region (e.g., 240) of a sequestrationpen can be, for example, at least 5×10⁵, 8×10⁵, 1×10⁶, 2×10⁶, 4×10⁶,6×10⁶, 8×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸, or 8×10⁸ cubic microns, ormore. In various embodiments of microfluidic devices havingsequestration pens, the volume of a sequestration pen may be about5×10⁵, 6×10⁵, 8×10⁵, 1×10⁶, 2×10⁶, 4×10⁶, 8×10⁶, 1×10⁷, 3×10⁷, 5×10⁷, orabout 8×10⁷ cubic microns, or more. In some other embodiments, thevolume of a sequestration pen may be about 1 nanoliter to about 50nanoliters, 2 nanoliters to about 25 nanoliters, 2 nanoliters to about20 nanoliters, about 2 nanoliters to about 15 nanoliters, or about 2nanoliters to about 10 nanoliters.

In various embodiment, the microfluidic device has sequestration pensconfigured as in any of the embodiments discussed herein where themicrofluidic device has about 5 to about 10 sequestration pens, about 10to about 50 sequestration pens, about 100 to about 500 sequestrationpens; about 200 to about 1000 sequestration pens, about 500 to about1500 sequestration pens, about 1000 to about 2000 sequestration pens, orabout 1000 to about 3500 sequestration pens. The sequestration pens neednot all be the same size and may include a variety of configurations(e.g., different widths, different features within the sequestrationpen.

FIG. 2G illustrates a microfluidic device 280 according to oneembodiment. The microfluidic device 280 is illustrated in FIG. 2G is astylized diagram of a microfluidic device 100. In practice themicrofluidic device 280 and its constituent circuit elements (e.g.channels 122 and sequestration pens 128) would have the dimensionsdiscussed herein. The microfluidic circuit 120 illustrated in FIG. 2Ghas two ports 107, four distinct channels 122 and four distinct flowpaths 106. The microfluidic device 280 further comprises a plurality ofsequestration pens opening off of each channel 122. In the microfluidicdevice illustrated in FIG. 2G, the sequestration pens have a geometrysimilar to the pens illustrated in FIG. 2C and thus, have bothconnection regions and isolation regions. Accordingly, the microfluidiccircuit 120 includes both swept regions (e.g. channels 122 and portionsof the connection regions 236 within the maximum penetration depth D_(p)of the secondary flow 244) and non-swept regions (e.g. isolation regions240 and portions of the connection regions 236 not within the maximumpenetration depth D_(p) of the secondary flow 244).

FIGS. 3A through 3B shows various embodiments of system 150 which can beused to operate and observe microfluidic devices (e.g. 100, 200, 230,250, 280, 290, 320, 400, 450, 500, 700) according to the presentdisclosure. As illustrated in FIG. 3A, the system 150 can include astructure (“nest”) 300 configured to hold a microfluidic device 100 (notshown), or any other microfluidic device described herein. The nest 300can include a socket 302 capable of interfacing with the microfluidicdevice 320 (e.g., an optically-actuated electrokinetic device 100) andproviding electrical connections from power source 192 to microfluidicdevice 320. The nest 300 can further include an integrated electricalsignal generation subsystem 304. The electrical signal generationsubsystem 304 can be configured to supply a biasing voltage to socket302 such that the biasing voltage is applied across a pair of electrodesin the microfluidic device 320 when it is being held by socket 302.Thus, the electrical signal generation subsystem 304 can be part ofpower source 192. The ability to apply a biasing voltage to microfluidicdevice 320 does not mean that a biasing voltage will be applied at alltimes when the microfluidic device 320 is held by the socket 302.Rather, in most cases, the biasing voltage will be appliedintermittently, e.g., only as needed to facilitate the generation ofelectrokinetic forces, such as dielectrophoresis or electro-wetting, inthe microfluidic device 320.

As illustrated in FIG. 3A, the nest 300 can include a printed circuitboard assembly (PCBA) 322. The electrical signal generation subsystem304 can be mounted on and electrically integrated into the PCBA 322. Theexemplary support includes socket 302 mounted on PCBA 322, as well.

Typically, the electrical signal generation subsystem 304 will include awaveform generator (not shown). The electrical signal generationsubsystem 304 can further include an oscilloscope (not shown) and/or awaveform amplification circuit (not shown) configured to amplify awaveform received from the waveform generator. The oscilloscope, ifpresent, can be configured to measure the waveform supplied to themicrofluidic device 320 held by the socket 302. In certain embodiments,the oscilloscope measures the waveform at a location proximal to themicrofluidic device 320 (and distal to the waveform generator), thusensuring greater accuracy in measuring the waveform actually applied tothe device. Data obtained from the oscilloscope measurement can be, forexample, provided as feedback to the waveform generator, and thewaveform generator can be configured to adjust its output based on suchfeedback. An example of a suitable combined waveform generator andoscilloscope is the Red Pitaya™.

In certain embodiments, the nest 300 further comprises a controller 308,such as a microprocessor used to sense and/or control the electricalsignal generation subsystem 304. Examples of suitable microprocessorsinclude the Arduino™ microprocessors, such as the Arduino Nano™. Thecontroller 308 may be used to perform functions and analysis or maycommunicate with an external master controller 154 (shown in FIG. 1A) toperform functions and analysis. In the embodiment illustrated in FIG. 3Athe controller 308 communicates with a master controller 154 through aninterface 310 (e.g., a plug or connector).

In some embodiments, the nest 300 can comprise an electrical signalgeneration subsystem 304 comprising a Red Pitaya™ waveformgenerator/oscilloscope unit (“Red Pitaya unit”) and a waveformamplification circuit that amplifies the waveform generated by the RedPitaya unit and passes the amplified voltage to the microfluidic device100. In some embodiments, the Red Pitaya unit is configured to measurethe amplified voltage at the microfluidic device 320 and then adjust itsown output voltage as needed such that the measured voltage at themicrofluidic device 320 is the desired value. In some embodiments, thewaveform amplification circuit can have a +6.5V to −6.5V power supplygenerated by a pair of DC-DC converters mounted on the PCBA 322,resulting in a signal of up to 13 Vpp at the microfluidic device 100.

As illustrated in FIG. 3A, the support structure 300 (e.g., nest) canfurther include a thermal control subsystem 306. The thermal controlsubsystem 306 can be configured to regulate the temperature ofmicrofluidic device 320 held by the support structure 300. For example,the thermal control subsystem 306 can include a Peltier thermoelectricdevice (not shown) and a cooling unit (not shown). The Peltierthermoelectric device can have a first surface configured to interfacewith at least one surface of the microfluidic device 320. The coolingunit can be, for example, a cooling block (not shown), such as aliquid-cooled aluminum block. A second surface of the Peltierthermoelectric device (e.g., a surface opposite the first surface) canbe configured to interface with a surface of such a cooling block. Thecooling block can be connected to a fluidic path 314 configured tocirculate cooled fluid through the cooling block. In the embodimentillustrated in FIG. 3A, the support structure 300 comprises an inlet 316and an outlet 318 to receive cooled fluid from an external reservoir(not shown), introduce the cooled fluid into the fluidic path 314 andthrough the cooling block, and then return the cooled fluid to theexternal reservoir. In some embodiments, the Peltier thermoelectricdevice, the cooling unit, and/or the fluidic path 314 can be mounted ona casing 312 of the support structure 300. In some embodiments, thethermal control subsystem 306 is configured to regulate the temperatureof the Peltier thermoelectric device so as to achieve a targettemperature for the microfluidic device 320. Temperature regulation ofthe Peltier thermoelectric device can be achieved, for example, by athermoelectric power supply, such as a Pololu™ thermoelectric powersupply (Pololu Robotics and Electronics Corp.). The thermal controlsubsystem 306 can include a feedback circuit, such as a temperaturevalue provided by an analog circuit. Alternatively, the feedback circuitcan be provided by a digital circuit.

In some embodiments, the nest 300 can include a thermal controlsubsystem 306 with a feedback circuit that is an analog voltage dividercircuit (not shown) which includes a resistor (e.g., with resistance 1kOhm+/−0.1%, temperature coefficient +/−0.02 ppm/C0) and a NTCthermistor (e.g., with nominal resistance 1 kOhm+/−0.01%). In someinstances, the thermal control subsystem 306 measures the voltage fromthe feedback circuit and then uses the calculated temperature value asinput to an on-board PID control loop algorithm. Output from the PIDcontrol loop algorithm can drive, for example, both a directional and apulse-width-modulated signal pin on a Pololu™ motor drive (not shown) toactuate the thermoelectric power supply, thereby controlling the Peltierthermoelectric device.

The nest 300 can include a serial port 324 which allows themicroprocessor of the controller 308 to communicate with an externalmaster controller 154 via the interface 310 (not shown). In addition,the microprocessor of the controller 308 can communicate (e.g., via aPlink tool (not shown)) with the electrical signal generation subsystem304 and thermal control subsystem 306. Thus, via the combination of thecontroller 308, the interface 310, and the serial port 324, theelectrical signal generation subsystem 304 and the thermal controlsubsystem 306 can communicate with the external master controller 154.In this manner, the master controller 154 can, among other things,assist the electrical signal generation subsystem 304 by performingscaling calculations for output voltage adjustments. A Graphical UserInterface (GUI) (not shown) provided via a display device 170 coupled tothe external master controller 154, can be configured to plottemperature and waveform data obtained from the thermal controlsubsystem 306 and the electrical signal generation subsystem 304,respectively. Alternatively, or in addition, the GUI can allow forupdates to the controller 308, the thermal control subsystem 306, andthe electrical signal generation subsystem 304.

As discussed above, system 150 can include an imaging device 194. Insome embodiments, the imaging device 194 comprises a light modulatingsubsystem 330 (See FIG. 3B). The light modulating subsystem 330 caninclude a digital mirror device (DMD) or a microshutter array system(MSA), either of which can be configured to receive light from a lightsource 332 and transmits a subset of the received light into an opticaltrain of microscope 350. Alternatively, the light modulating subsystem330 can include a device that produces its own light (and thus dispenseswith the need for a light source 332), such as an organic light emittingdiode display (OLED), a liquid crystal on silicon (LCOS) device, aferroelectric liquid crystal on silicon device (FLCOS), or atransmissive liquid crystal display (LCD). The light modulatingsubsystem 330 can be, for example, a projector. Thus, the lightmodulating subsystem 330 can be capable of emitting both structured andunstructured light. One example of a suitable light modulating subsystem330 is the Mosaic™ system from Andor Technologies™. In certainembodiments, imaging module 164 and/or motive module 162 of system 150can control the light modulating subsystem 330.

In certain embodiments, the imaging device 194 further comprises amicroscope 350. In such embodiments, the nest 300 and light modulatingsubsystem 330 can be individually configured to be mounted on themicroscope 350. The microscope 350 can be, for example, a standardresearch-grade light microscope or fluorescence microscope. Thus, thenest 300 can be configured to be mounted on the stage 344 of themicroscope 350 and/or the light modulating subsystem 330 can beconfigured to mount on a port of microscope 350. In other embodiments,the nest 300 and the light modulating subsystem 330 described herein canbe integral components of microscope 350.

In certain embodiments, the microscope 350 can further include one ormore detectors 348. In some embodiments, the detector 348 is controlledby the imaging module 164. The detector 348 can include an eye piece, acharge-coupled device (CCD), a camera (e.g., a digital camera), or anycombination thereof. If at least two detectors 348 are present, onedetector can be, for example, a fast-frame-rate camera while the otherdetector can be a high sensitivity camera. Furthermore, the microscope350 can include an optical train configured to receive reflected and/oremitted light from the microfluidic device 320 and focus at least aportion of the reflected and/or emitted light on the one or moredetectors 348. The optical train of the microscope can also includedifferent tube lenses (not shown) for the different detectors, such thatthe final magnification on each detector can be different.

In certain embodiments, imaging device 194 is configured to use at leasttwo light sources. For example, a first light source 332 can be used toproduce structured light (e.g., via the light modulating subsystem 330)and a second light source 334 can be used to provide unstructured light.The first light source 332 can produce structured light foroptically-actuated electrokinesis and/or fluorescent excitation, and thesecond light source 334 can be used to provide bright fieldillumination. In these embodiments, the motive module 164 can be used tocontrol the first light source 332 and the imaging module 164 can beused to control the second light source 334. The optical train of themicroscope 350 can be configured to (1) receive structured light fromthe light modulating subsystem 330 and focus the structured light on atleast a first region in a microfluidic device, such as anoptically-actuated electrokinetic device, when the device is being heldby the nest 300, and (2) receive reflected and/or emitted light from themicrofluidic device and focus at least a portion of such reflectedand/or emitted light onto detector 348. The optical train can be furtherconfigured to receive unstructured light from a second light source andfocus the unstructured light on at least a second region of themicrofluidic device, when the device is held by the nest 300. In certainembodiments, the first and second regions of the microfluidic device canbe overlapping regions. For example, the first region can be a subset ofthe second region.

In FIG. 3B, the first light source 332 is shown supplying light to alight modulating subsystem 330, which provides structured light to theoptical train of the microscope 350 of system 355 (not shown). Thesecond light source 334 is shown providing unstructured light to theoptical train via a beam splitter 336. Structured light from the lightmodulating subsystem 330 and unstructured light from the second lightsource 334 travel from the beam splitter 336 through the optical traintogether to reach a second beam splitter (or dichroic filter 338,depending on the light provided by the light modulating subsystem 330),where the light gets reflected down through the objective 336 to thesample plane 342. Reflected and/or emitted light from the sample plane342 then travels back up through the objective 340, through the beamsplitter and/or dichroic filter 338, and to a dichroic filter 346. Onlya fraction of the light reaching dichroic filter 346 passes through andreaches the detector 348.

In some embodiments, the second light source 334 emits blue light. Withan appropriate dichroic filter 346, blue light reflected from the sampleplane 342 is able to pass through dichroic filter 346 and reach thedetector 348. In contrast, structured light coming from the lightmodulating subsystem 330 gets reflected from the sample plane 342, butdoes not pass through the dichroic filter 346. In this example, thedichroic filter 346 is filtering out visible light having a wavelengthlonger than 495 nm. Such filtering out of the light from the lightmodulating subsystem 330 would only be complete (as shown) if the lightemitted from the light modulating subsystem did not include anywavelengths shorter than 495 nm. In practice, if the light coming fromthe light modulating subsystem 330 includes wavelengths shorter than 495nm (e.g., blue wavelengths), then some of the light from the lightmodulating subsystem would pass through filter 346 to reach the detector348. In such an embodiment, the filter 346 acts to change the balancebetween the amount of light that reaches the detector 348 from the firstlight source 332 and the second light source 334. This can be beneficialif the first light source 332 is significantly stronger than the secondlight source 334. In other embodiments, the second light source 334 canemit red light, and the dichroic filter 346 can filter out visible lightother than red light (e.g., visible light having a wavelength shorterthan 650 nm).

Coating Solutions and Coating Agents.

Without intending to be limited by theory, maintenance of a biologicalmicro-object (e.g., a biological cell) within a microfluidic device(e.g., a DEP-configured and/or EW-configured microfluidic device) may befacilitated (i.e., the biological micro-object exhibits increasedviability, greater expansion and/or greater portability within themicrofluidic device) when at least one or more inner surfaces of themicrofluidic device have been conditioned or coated so as to present alayer of organic and/or hydrophilic molecules that provides the primaryinterface between the microfluidic device and biological micro-object(s)maintained therein. In some embodiments, one or more of the innersurfaces of the microfluidic device (e.g. the inner surface of theelectrode activation substrate of a DEP-configured microfluidic device,the cover of the microfluidic device, and/or the surfaces of the circuitmaterial) may be treated with or modified by a coating solution and/orcoating agent to generate the desired layer of organic and/orhydrophilic molecules.

The coating may be applied before or after introduction of biologicalmicro-object(s), or may be introduced concurrently with the biologicalmicro-object(s). In some embodiments, the biological micro-object(s) maybe imported into the microfluidic device in a fluidic medium thatincludes one or more coating agents. In other embodiments, the innersurface(s) of the microfluidic device (e.g., a DEP-configuredmicrofluidic device) are treated or “primed” with a coating solutioncomprising a coating agent prior to introduction of the biologicalmicro-object(s) into the microfluidic device.

In some embodiments, at least one surface of the microfluidic deviceincludes a coating material that provides a layer of organic and/orhydrophilic molecules suitable for maintenance and/or expansion ofbiological micro-object(s) (e.g. provides a conditioned surface asdescribed below). In some embodiments, substantially all the innersurfaces of the microfluidic device include the coating material. Thecoated inner surface(s) may include the surface of a flow region (e.g.,channel), chamber, or sequestration pen, or a combination thereof. Insome embodiments, each of a plurality of sequestration pens has at leastone inner surface coated with coating materials. In other embodiments,each of a plurality of flow regions or channels has at least one innersurface coated with coating materials. In some embodiments, at least oneinner surface of each of a plurality of sequestration pens and each of aplurality of channels is coated with coating materials.

Coating Agent/Solution.

Any convenient coating agent/coating solution can be used, including butnot limited to: serum or serum factors, bovine serum albumin (BSA),polymers, detergents, enzymes, and any combination thereof.

Polymer-Based Coating Materials.

The at least one inner surface may include a coating material thatcomprises a polymer. The polymer may be covalently or non-covalentlybound (or may be non-specifically adhered) to the at least one surface.The polymer may have a variety of structural motifs, such as found inblock polymers (and copolymers), star polymers (star copolymers), andgraft or comb polymers (graft copolymers), all of which may be suitablefor the methods disclosed herein.

The polymer may include a polymer including alkylene ether moieties. Awide variety of alkylene ether containing polymers may be suitable foruse in the microfluidic devices described herein. One non-limitingexemplary class of alkylene ether containing polymers are amphiphilicnonionic block copolymers which include blocks of polyethylene oxide(PEO) and polypropylene oxide (PPO) subunits in differing ratios andlocations within the polymer chain. Pluronic® polymers (BASF) are blockcopolymers of this type and are known in the art to be suitable for usewhen in contact with living cells. The polymers may range in averagemolecular mass M_(w) from about 2000 Da to about 20 KDa. In someembodiments, the PEO-PPO block copolymer can have ahydrophilic-lipophilic balance (HLB) greater than about 10 (e.g. 12-18).Specific Pluronic® polymers useful for yielding a coated surface includePluronic® L44, L64, P85, and F127 (including F127NF). Another class ofalkylene ether containing polymers is polyethylene glycol (PEGM_(w)<100,000 Da) or alternatively polyethylene oxide (PEO,M_(w)>100,000). In some embodiments, a PEG may have an M_(w) of about1000 Da, 5000 Da, 10,000 Da or 20,000 Da.

In other embodiments, the coating material may include a polymercontaining carboxylic acid moieties. The carboxylic acid subunit may bean alkyl, alkenyl or aromatic moiety containing subunit. Onenon-limiting example is polylactic acid (PLA). In other embodiments, thecoating material may include a polymer containing phosphate moieties,either at a terminus of the polymer backbone or pendant from thebackbone of the polymer. In yet other embodiments, the coating materialmay include a polymer containing sulfonic acid moieties. The sulfonicacid subunit may be an alkyl, alkenyl or aromatic moiety containingsubunit. One non-limiting example is polystyrene sulfonic acid (PSSA) orpolyanethole sulfonic acid. In further embodiments, the coating materialmay include a polymer including amine moieties. The polyamino polymermay include a natural polyamine polymer or a synthetic polyaminepolymer. Examples of natural polyamines include spermine, spermidine,and putrescine.

In other embodiments, the coating material may include a polymercontaining saccharide moieties. In a non-limiting example,polysaccharides such as xanthan gum or dextran may be suitable to form amaterial which may reduce or prevent cell sticking in the microfluidicdevice. For example, a dextran polymer having a size about 3 kDa may beused to provide a coating material for a surface within a microfluidicdevice.

In other embodiments, the coating material may include a polymercontaining nucleotide moieties, i.e. a nucleic acid, which may haveribonucleotide moieties or deoxyribonucleotide moieties, providing apolyelectrolyte surface. The nucleic acid may contain only naturalnucleotide moieties or may contain unnatural nucleotide moieties whichcomprise nucleobase, ribose or phosphate moiety analogs such as7-deazaadenine, pentose, methyl phosphonate or phosphorothioate moietieswithout limitation.

In yet other embodiments, the coating material may include a polymercontaining amino acid moieties. The polymer containing amino acidmoieties may include a natural amino acid containing polymer or anunnatural amino acid containing polymer, either of which may include apeptide, a polypeptide or a protein. In one non-limiting example, theprotein may be bovine serum albumin (BSA) and/or serum (or a combinationof multiple different sera) comprising albumin and/or one or more othersimilar proteins as coating agents. The serum can be from any convenientsource, including but not limited to fetal calf serum, sheep serum, goatserum, horse serum, and the like. In certain embodiments, BSA in acoating solution is present in a range of form about 1 mg/mL to about100 mg/mL, including 5 mg/mL, 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, 50mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, or more or anywhere inbetween. In certain embodiments, serum in a coating solution may bepresent in a range of from about 20% (v/v) to about 50% v/v, including25%, 30%, 35%, 40%, 45%, or more or anywhere in between. In someembodiments, BSA may be present as a coating agent in a coating solutionat 5 mg/mL, whereas in other embodiments, BSA may be present as acoating agent in a coating solution at 70 mg/mL. In certain embodiments,serum is present as a coating agent in a coating solution at 30%. Insome embodiments, an extracellular matrix (ECM) protein may be providedwithin the coating material for optimized cell adhesion to foster cellgrowth. A cell matrix protein, which may be included in a coatingmaterial, can include, but is not limited to, a collagen, an elastin, anRGD-containing peptide (e.g. a fibronectin), or a laminin. In yet otherembodiments, growth factors, cytokines, hormones or other cell signalingspecies may be provided within the coating material of the microfluidicdevice.

In some embodiments, the coating material may include a polymercontaining more than one of alkylene oxide moieties, carboxylic acidmoieties, sulfonic acid moieties, phosphate moieties, saccharidemoieties, nucleotide moieties, or amino acid moieties. In otherembodiments, the polymer conditioned surface may include a mixture ofmore than one polymer each having alkylene oxide moieties, carboxylicacid moieties, sulfonic acid moieties, phosphate moieties, saccharidemoieties, nucleotide moieties, and/or amino acid moieties, which may beindependently or simultaneously incorporated into the coating material.

Covalently Linked Coating Materials.

In some embodiments, the at least one inner surface includes covalentlylinked molecules that provide a layer of organic and/or hydrophilicmolecules suitable for maintenance/expansion of biologicalmicro-object(s) within the microfluidic device, providing a conditionedsurface for such cells.

The covalently linked molecules include a linking group, wherein thelinking group is covalently linked to one or more surfaces of themicrofluidic device, as described below. The linking group is alsocovalently linked to a moiety configured to provide a layer of organicand/or hydrophilic molecules suitable for maintenance/expansion ofbiological micro-object(s).

In some embodiments, the covalently linked moiety configured to providea layer of organic and/or hydrophilic molecules suitable formaintenance/expansion of biological micro-object(s) may include alkyl orfluoroalkyl (which includes perfluoroalkyl) moieties; mono- orpolysaccharides (which may include but is not limited to dextran);alcohols (including but not limited to propargyl alcohol); polyalcohols,including but not limited to polyvinyl alcohol; alkylene ethers,including but not limited to polyethylene glycol; polyelectrolytes(including but not limited to polyacrylic acid or polyvinyl phosphonicacid); amino groups (including derivatives thereof, such as, but notlimited to alkylated amines, hydroxyalkylated amino group, guanidinium,and heterocylic groups containing an unaromatized nitrogen ring atom,such as, but not limited to morpholinyl or piperazinyl); carboxylicacids including but not limited to propiolic acid (which may provide acarboxylate anionic surface); phosphonic acids, including but notlimited to ethynyl phosphonic acid (which may provide a phosphonateanionic surface); sulfonate anions; carboxybetaines; sulfobetaines;sulfamic acids; or amino acids.

In various embodiments, the covalently linked moiety configured toprovide a layer of organic and/or hydrophilic molecules suitable formaintenance/expansion of biological micro-object(s) in the microfluidicdevice may include non-polymeric moieties such as an alkyl moiety, asubstituted alkyl moiety, such as a fluoroalkyl moiety (including butnot limited to a perfluoroalkyl moiety), amino acid moiety, alcoholmoiety, amino moiety, carboxylic acid moiety, phosphonic acid moiety,sulfonic acid moiety, sulfamic acid moiety, or saccharide moiety.Alternatively, the covalently linked moiety may include polymericmoieties, which may be any of the moieties described above.

In some embodiments, the covalently linked alkyl moiety may comprisescarbon atoms forming a linear chain (e.g., a linear chain of at least 10carbons, or at least 14, 16, 18, 20, 22, or more carbons) and may be anunbranched alkyl moiety. In some embodiments, the alkyl group mayinclude a substituted alkyl group (e.g., some of the carbons in thealkyl group can be fluorinated or perfluorinated). In some embodiments,the alkyl group may include a first segment, which may include aperfluoroalkyl group, joined to a second segment, which may include anon-substituted alkyl group, where the first and second segments may bejoined directly or indirectly (e.g., by means of an ether linkage). Thefirst segment of the alkyl group may be located distal to the linkinggroup, and the second segment of the alkyl group may be located proximalto the linking group.

In other embodiments, the covalently linked moiety may include at leastone amino acid, which may include more than one type of amino acid.Thus, the covalently linked moiety may include a peptide or a protein.In some embodiments, the covalently linked moiety may include an aminoacid which may provide a zwitterionic surface to support cell growth,viability, portability, or any combination thereof.

In other embodiments, the covalently linked moiety may include at leastone alkylene oxide moiety, and may include any alkylene oxide polymer asdescribed above. One useful class of alkylene ether containing polymersis polyethylene glycol (PEG M_(w)<100,000 Da) or alternativelypolyethylene oxide (PEO, M_(w)>100,000). In some embodiments, a PEG mayhave an M_(w) of about 1000 Da, 5000 Da, 10,000 Da or 20,000 Da.

The covalently linked moiety may include one or more saccharides. Thecovalently linked saccharides may be mono-, di-, or polysaccharides. Thecovalently linked saccharides may be modified to introduce a reactivepairing moiety which permits coupling or elaboration for attachment tothe surface. Exemplary reactive pairing moieties may include aldehyde,alkyne or halo moieties. A polysaccharide may be modified in a randomfashion, wherein each of the saccharide monomers may be modified or onlya portion of the saccharide monomers within the polysaccharide aremodified to provide a reactive pairing moiety that may be coupleddirectly or indirectly to a surface. One exemplar may include a dextranpolysaccharide, which may be coupled indirectly to a surface via anunbranched linker.

The covalently linked moiety may include one or more amino groups. Theamino group may be a substituted amine moiety, guanidine moiety,nitrogen-containing heterocyclic moiety or heteroaryl moiety. The aminocontaining moieties may have structures permitting pH modification ofthe environment within the microfluidic device, and optionally, withinthe sequestration pens and/or flow regions (e.g., channels).

The coating material providing a conditioned surface may comprise onlyone kind of covalently linked moiety or may include more than onedifferent kind of covalently linked moiety. For example, the fluoroalkylconditioned surfaces (including perfluoroalkyl) may have a plurality ofcovalently linked moieties which are all the same, e.g., having the samelinking group and covalent attachment to the surface, the same overalllength, and the same number of fluoromethylene units comprising thefluoroalkyl moiety. Alternatively, the coating material may have morethan one kind of covalently linked moiety attached to the surface. Forexample, the coating material may include molecules having covalentlylinked alkyl or fluoroalkyl moieties having a specified number ofmethylene or fluoromethylene units and may further include a further setof molecules having charged moieties covalently attached to an alkyl orfluoroalkyl chain having a greater number of methylene orfluoromethylene units, which may provide capacity to present bulkiermoieties at the coated surface. In this instance, the first set ofmolecules having different, less sterically demanding termini and fewerbackbone atoms can help to functionalize the entire substrate surfaceand thereby prevent undesired adhesion or contact with thesilicon/silicon oxide, hafnium oxide or alumina making up the substrateitself. In another example, the covalently linked moieties may provide azwitterionic surface presenting alternating charges in a random fashionon the surface.

Conditioned Surface Properties.

Aside from the composition of the conditioned surface, other factorssuch as physical thickness of the hydrophobic material can impact DEPforce. Various factors can alter the physical thickness of theconditioned surface, such as the manner in which the conditioned surfaceis formed on the substrate (e.g. vapor deposition, liquid phasedeposition, spin coating, flooding, and electrostatic coating). In someembodiments, the conditioned surface has a thickness in the range ofabout 1 nm to about 10 nm; about 1 nm to about 7 nm; about 1 nm to about5 nm; or any individual value therebetween. In other embodiments, theconditioned surface formed by the covalently linked moieties may have athickness of about 10 nm to about 50 nm. In various embodiments, theconditioned surface prepared as described herein has a thickness of lessthan 10 nm. In some embodiments, the covalently linked moieties of theconditioned surface may form a monolayer when covalently linked to thesurface of the microfluidic device (e.g., a DEP configured substratesurface) and may have a thickness of less than 10 nm (e.g., less than 5nm, or about 1.5 to 3.0 nm). These values are in contrast to that of aCYTOP® (Asahi Glass Co., Ltd. JP) fluoropolymer spin coating, which hasa thickness in the range of about 30 nm. In some embodiments, theconditioned surface does not require a perfectly formed monolayer to besuitably functional for operation within a DEP-configured microfluidicdevice.

In various embodiments, the coating material providing a conditionedsurface of the microfluidic device may provide desirable electricalproperties. Without intending to be limited by theory, one factor thatimpacts robustness of a surface coated with a particular coatingmaterial is intrinsic charge trapping. Different coating materials maytrap electrons, which can lead to breakdown of the coating material.Defects in the coating material may increase charge trapping and lead tofurther breakdown of the coating material. Similarly, different coatingmaterials have different dielectric strengths (i.e. the minimum appliedelectric field that results in dielectric breakdown), which may impactcharge trapping. In certain embodiments, the coating material can havean overall structure (e.g., a densely-packed monolayer structure) thatreduces or limits that amount of charge trapping.

In addition to its electrical properties, the conditioned surface mayalso have properties that are beneficial in use with biologicalmolecules. For example, a conditioned surface that contains fluorinated(or perfluorinated) carbon chains may provide a benefit relative toalkyl-terminated chains in reducing the amount of surface fouling.Surface fouling, as used herein, refers to the amount of indiscriminatematerial deposition on the surface of the microfluidic device, which mayinclude permanent or semi-permanent deposition of biomaterials such asprotein and its degradation products, nucleic acids and respectivedegradation products and the like.

Unitary or Multi-Part Conditioned Surface.

The covalently linked coating material may be formed by reaction of amolecule which already contains the moiety configured to provide a layerof organic and/or hydrophilic molecules suitable formaintenance/expansion of biological micro-object(s) in the microfluidicdevice, as is described below. Alternatively, the covalently linkedcoating material may be formed in a two-part sequence by coupling themoiety configured to provide a layer of organic and/or hydrophilicmolecules suitable for maintenance/expansion of biologicalmicro-object(s) to a surface modifying ligand that itself has beencovalently linked to the surface.

Methods of Preparing a Covalently Linked Coating Material.

In some embodiments, a coating material that is covalently linked to thesurface of a microfluidic device (e.g., including at least one surfaceof the sequestration pens and/or flow regions) has a structure ofFormula 1 or Formula 2. When the coating material is introduced to thesurface in one step, it has a structure of Formula 1, while when thecoating material is introduced in a multiple step process, it has astructure of Formula 2.

The coating material may be linked covalently to oxides of the surfaceof a DEP-configured or EW-configured substrate. The DEP- orEW-configured substrate may comprise silicon, silicon oxide, alumina, orhafnium oxide. Oxides may be present as part of the native chemicalstructure of the substrate or may be introduced as discussed below.

The coating material may be attached to the oxides via a linking group(“LG”), which may be a siloxy or phosphonate ester group formed from thereaction of a siloxane or phosphonic acid group with the oxides. Themoiety configured to provide a layer of organic and/or hydrophilicmolecules suitable for maintenance/expansion of biologicalmicro-object(s) in the microfluidic device can be any of the moietiesdescribed herein. The linking group LG may be directly or indirectlyconnected to the moiety configured to provide a layer of organic and/orhydrophilic molecules suitable for maintenance/expansion of biologicalmicro-object(s) in the microfluidic device. When the linking group LG isdirectly connected to the moiety, optional linker (“L”) is not presentand n is 0. When the linking group LG is indirectly connected to themoiety, linker L is present and n is 1. The linker L may have a linearportion where a backbone of the linear portion may include 1 to 200non-hydrogen atoms selected from any combination of silicon, carbon,nitrogen, oxygen, sulfur and phosphorus atoms, subject to chemicalbonding limitations as is known in the art. It may be interrupted withany combination of one or more moieties selected from the groupconsisting of ether, amino, carbonyl, amido, or phosphonate groups,arylene, heteroarylene, or heterocyclic groups. In some embodiments, thebackbone of the linker L may include 10 to 20 atoms. In otherembodiments, the backbone of the linker L may include about 5 atoms toabout 200 atoms; about 10 atoms to about 80 atoms; about 10 atoms toabout 50 atoms; or about 10 atoms to about 40 atoms. In someembodiments, the backbone atoms are all carbon atoms.

In some embodiments, the moiety configured to provide a layer of organicand/or hydrophilic molecules suitable for maintenance/expansion ofbiological micro-object(s) may be added to the surface of the substratein a multi-step process, and has a structure of Formula 2, as shownabove. The moiety may be any of the moieties described above.

In some embodiments, the coupling group CG represents the resultantgroup from reaction of a reactive moiety R_(x) and a reactive pairingmoiety R_(px) (i.e., a moiety configured to react with the reactivemoiety R_(x)). For example, one typical coupling group CG may include acarboxamidyl group, which is the result of the reaction of an aminogroup with a derivative of a carboxylic acid, such as an activatedester, an acid chloride or the like. Other CG may include a triazolylenegroup, a carboxamidyl, thioamidyl, an oxime, a mercaptyl, a disulfide,an ether, or alkenyl group, or any other suitable group that may beformed upon reaction of a reactive moiety with its respective reactivepairing moiety. The coupling group CG may be located at the second end(i.e., the end proximal to the moiety configured to provide a layer oforganic and/or hydrophilic molecules suitable for maintenance/expansionof biological micro-object(s) in the microfluidic device) of linker L,which may include any combination of elements as described above. Insome other embodiments, the coupling group CG may interrupt the backboneof the linker L. When the coupling group CG is triazolylene, it may bethe product resulting from a Click coupling reaction and may be furthersubstituted (e.g., a dibenzocylcooctenyl fused triazolylene group).

In some embodiments, the coating material (or surface modifying ligand)is deposited on the inner surfaces of the microfluidic device usingchemical vapor deposition. The vapor deposition process can beoptionally improved, for example, by pre-cleaning the cover 110, themicrofluidic circuit material 116, and/or the substrate (e.g., the innersurface 208 of the electrode activation substrate 206 of aDEP-configured substrate, or a dielectric layer of the support structure104 of an EW-configured substrate), by exposure to a solvent bath,sonication or a combination thereof. Alternatively, or in addition, suchpre-cleaning can include treating the cover 110, the microfluidiccircuit material 116, and/or the substrate in an oxygen plasma cleaner,which can remove various impurities, while at the same time introducingan oxidized surface (e.g. oxides at the surface, which may be covalentlymodified as described herein). Alternatively, liquid-phase treatments,such as a mixture of hydrochloric acid and hydrogen peroxide or amixture of sulfuric acid and hydrogen peroxide (e.g., piranha solution,which may have a ratio of sulfuric acid to hydrogen peroxide in a rangefrom about 3:1 to about 7:1) may be used in place of an oxygen plasmacleaner.

In some embodiments, vapor deposition is used to coat the inner surfacesof the microfluidic device 200 after the microfluidic device 200 hasbeen assembled to form an enclosure 102 defining a microfluidic circuit120. Without intending to be limited by theory, depositing such acoating material on a fully-assembled microfluidic circuit 120 may bebeneficial in preventing delamination caused by a weakened bond betweenthe microfluidic circuit material 116 and the electrode activationsubstrate 206 dielectric layer and/or the cover 110. In embodimentswhere a two-step process is employed the surface modifying ligand may beintroduced via vapor deposition as described above, with subsequentintroduction of the moiety configured provide a layer of organic and/orhydrophilic molecules suitable for maintenance/expansion of biologicalmicro-object(s). The subsequent reaction may be performed by exposingthe surface modified microfluidic device to a suitable coupling reagentin solution.

FIG. 2H depicts a cross-sectional views of a microfluidic device 290having an exemplary covalently linked coating material providing aconditioned surface. As illustrated, the coating materials 298 (shownschematically) can comprise a monolayer of densely-packed moleculescovalently bound to both the inner surface 294 of a base 286, which maybe a DEP substrate, and the inner surface 292 of a cover 288 of themicrofluidic device 290. The coating material 298 can be disposed onsubstantially all inner surfaces 294, 292 proximal to, and facinginwards towards, the enclosure 284 of the microfluidic device 290,including, in some embodiments and as discussed above, the surfaces ofmicrofluidic circuit material (not shown) used to define circuitelements and/or structures within the microfluidic device 290. Inalternate embodiments, the coating material 298 can be disposed on onlyone or some of the inner surfaces of the microfluidic device 290.

In the embodiment shown in FIG. 2H, the coating material 298 can includea monolayer of organosiloxane molecules, each molecule covalently bondedto the inner surfaces 292, 294 of the microfluidic device 290 via asiloxy linker 296. Any of the above-discussed coating materials 298 canbe used (e.g. an alkyl-terminated, a fluoroalkyl terminated moiety, aPEG-terminated moiety, a dextran terminated moiety, or a terminal moietycontaining positive or negative charges for the organosiloxy moieties),where the terminal moiety is disposed at its enclosure-facing terminus(i.e. the portion of the monolayer of the coating material 298 that isnot bound to the inner surfaces 292, 294 and is proximal to theenclosure 284).

In other embodiments, the coating material 298 used to coat the innersurface(s) 292, 294 of the microfluidic device 290 can include anionic,cationic, or zwitterionic moieties, or any combination thereof. Withoutintending to be limited by theory, by presenting cationic moieties,anionic moieties, and/or zwitterionic moieties at the inner surfaces ofthe enclosure 284 of the microfluidic circuit 120, the coating material298 can form strong hydrogen bonds with water molecules such that theresulting water of hydration acts as a layer (or “shield”) thatseparates the biological micro-objects from interactions withnon-biological molecules (e.g., the silicon and/or silicon oxide of thesubstrate). In addition, in embodiments in which the coating material298 is used in conjunction with coating agents, the anions, cations,and/or zwitterions of the coating material 298 can form ionic bonds withthe charged portions of non-covalent coating agents (e.g. proteins insolution) that are present in a medium 180 (e.g. a coating solution) inthe enclosure 284.

In still other embodiments, the coating material may comprise or bechemically modified to present a hydrophilic coating agent at itsenclosure-facing terminus. In some embodiments, the coating material mayinclude an alkylene ether containing polymer, such as PEG. In someembodiments, the coating material may include a polysaccharide, such asdextran. Like the charged moieties discussed above (e.g., anionic,cationic, and zwitterionic moieties), the hydrophilic coating agent canform strong hydrogen bonds with water molecules such that the resultingwater of hydration acts as a layer (or “shield”) that separates thebiological micro-objects from interactions with non-biological molecules(e.g., the silicon and/or silicon oxide of the substrate).

Further details of appropriate coating treatments and modifications maybe found at U.S. application Ser. No. 15/135,707, filed on Apr. 22,2016, and is incorporated by reference in its entirety.

Additional System Components for Maintenance of Viability of Cellswithin the Sequestration Pens of the Microfluidic Device.

In order to promote growth and/or expansion of cell populations,environmental conditions conducive to maintaining functional cells maybe provided by additional components of the system. For example, suchadditional components can provide nutrients, cell growth signalingspecies, pH modulation, gas exchange, temperature control, and removalof waste products from cells.

Recitation of Some Embodiments of the Microfluidic Devices, Methods andKits

1. A microfluidic device including: an enclosure including a substrateand a microfluidic circuit material, the enclosure defining a flowregion; and at least one in situ-generated capture structure disposedwithin the enclosure, and optionally within the flow region, where theat least one in situ-generated capture structure comprises a solidifiedpolymer network.

2. The microfluidic device of embodiment 1, where the solidified polymernetwork may include one or more functionalized sites.

3. The microfluidic device of embodiment 1 or 2, where the solidifiedpolymer network may include an assay reagent or assay analyte.

4. The microfluidic device of any one of embodiments 1-3, where theenclosure of the microfluidic device may include at least onesequestration pen, and optionally a proximal opening of thesequestration pen to the flow region may be oriented substantiallyparallel to an average direction of flow of fluidic medium in the flowregion.

5. The microfluidic device of embodiment 4, where the at least onesequestration pen may include an isolation region and a connectionregion, the connection region having a proximal opening to the flowregion and a distal opening to the isolation region.

6. The microfluidic device of embodiment 4 or 5, where the one or morein situ-generated capture structures may be disposed within thesequestration pen, and optionally within the isolation region of thesequestration pen.

7. The microfluidic device of any one of embodiments 3-6, where theassay reagent may be covalently attached to the solidified polymernetwork.

8. The microfluidic device of any one of embodiments 3-6, where theassay reagent may be non-covalently attached to the solidified polymernetwork.

9. The microfluidic device of embodiment 8, where the assay reagent maybe non-covalently attached to the solidified polymer network via abiotin/streptavidin complex.

10. The microfluidic device of any one of embodiments 3-9, where theassay reagent may be a protein, a nucleic acid, an organic molecule,and/or a saccharide.

11. The microfluidic device of embodiment 10, where the assay reagentmay include an antibody.

12. The microfluidic device of embodiment 10, where the assay reagentmay include an antigen.

13. The microfluidic device of embodiment 10, where the assay reagentmay include a capture oligonucleotide.

14. The microfluidic device of any one of embodiments 3-6, where theassay analyte may be non-covalently bound to the solidified polymernetwork of the at least one in situ-generated capture structure.

15. The microfluidic device of any one of embodiments 3-6 or 14, wherethe assay analyte may include a protein.

16. The microfluidic device of any one of embodiments 3-6 or 14, wherethe assay analyte may include an oligonucleotide.

17. The microfluidic device of any one of embodiments 3-6 or 14, wherethe assay analyte may include an antibody or a cytokine.

18. The microfluidic device of any one of embodiments 3-6 or 14, wherethe assay analyte may include an organic molecule.

19. The microfluidic device of any one of embodiments 1-18, where two ormore in situ-generated capture structures may be disposed in the flowregion and/or the at least one sequestration pen.

20. The microfluidic device of embodiment 19, where a first capturestructure of the two or more in situ-generated capture structures maybind to a first assay reagent or a first assay analyte, and a secondcapture structure of the two or more in situ-generated capturestructures may bind to a second assay reagent or a second assay analyte,where the first assay reagent or first assay analyte is different fromthe second assay reagent or second assay analyte.

21. The microfluidic device of embodiment 19 or 20, where each of thetwo or more in situ-generated capture structures may be disposed at adifferent location in the at least one sequestration pen.

22. The microfluidic device of any one of embodiments 1-21, where acover of the microfluidic device may be substantially transparent to afluorescent, colorimetric, or luminescent signal from the one or morecapture structures.

23. The microfluidic device of any one of embodiments 1-22, where thesolidified polymer network may include a photoinitiated polymer.

24. The microfluidic device of any one of embodiments 1-23, where thesolidified polymer network may include a synthetic polymer, a modifiedsynthetic polymer, a biological polymer, or any combination thereof.

25. The microfluidic device of embodiment 24, where the modifiedsynthetic polymer may include cleavage motifs, reactive terminalmoieties, and/or cell recognition motifs.

26. The microfluidic device of any one of embodiments 1-25, where thesolidified polymer network may include at least one of a polyethyleneglycol, modified polyethylene glycol, polylactic acid (PLA), modifiedpolylactic acid, polyglycolic acid (PGA), modified polyglycolic acid,polyacrylamide (PAM), modified polyacrylamide,poly-N-isopropylacrylamide (PNIPAm), modifiedpoly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinylalcohol, polyacrylic acid (PAA), modified polyacrylic acid,polycaprolactone (PCL), modified polycaprolactone, fibronectin, modifiedfibronectin, collagen, modified collagen, laminin, modified laminin,polysaccharide, modified polysaccharide, or any co-polymer combinationthereof.

27. The microfluidic device of any one of embodiments 1-26, wherein thesolidified polymer network comprises a modified polyethylene glycolpolymer.

28. The microfluidic device of any one of embodiments 1-27, where themicrofluidic device may include a plurality of sequestration pens.

29. The microfluidic device of any one of embodiments 4-28, where the atleast one in situ-generated capture structure may be configured topermit exit of a micro-object from the sequestration pen.

30. The microfluidic device of any one of embodiments 1-29, where thesubstrate may be configured to generate dielectrophoresis (DEP) forceswithin the enclosure.

31. The microfluidic device of embodiment 30, where the DEP forces maybe optically actuated.

32. The microfluidic device of any one of embodiments 1-31, where atleast one inner surface of the microfluidic device may further include aconditioned surface.

33. A method of assaying a micro-object in a microfluidic deviceincluding at least a first in situ-generated capture structure, themethod including: disposing a micro-object within a microfluidic devicein a region proximal to the at least first in situ-generated capturestructure, the in situ-generated capture structure including asolidified polymer network, where the solidified polymer networkcomprises an assay reagent or assay analyte; contacting the assayreagent or assay analyte with the micro-object or a biological productof the micro-object; and detecting an interaction of the assay reagentor assay analyte with the micro-object or the biological product.

34. The method of embodiment 33, where the microfluidic device mayinclude an enclosure including: a substrate; a flow region; and,optionally, at least one sequestration pen, where the first insitu-generated capture structure is disposed within the flow region orthe at least one sequestration pen.

35. The method of embodiments 33 or 34, where the at least onesequestration pen may include an isolation region and a connectionregion, the connection region having a proximal opening to the flowregion and a distal opening to the isolation region.

36. The method of embodiment 35, where the at least first insitu-generated capture structure may be disposed within the isolationregion of the sequestration pen.

37. The method of any one of embodiments 33-36, where the assay reagentor assay analyte may be non-covalently attached to the solidifiedpolymer network.

38. The method of any one of embodiments 33-37, where the assay reagentor assay analyte may include a protein, an oligonucleotide, an organicmolecule, or a saccharide.

39. The method of any one of embodiments 33-38, where the assay reagentmay include an antibody.

40. The method of any one of embodiments 33-39, where the biologicalproduct may include a protein, an oligonucleotide, an organic molecule,or a saccharide.

41. The method of embodiment 40, where the protein biological productmay include an antibody or an antigen.

42. The method of any one of embodiments 33-41, where the micro-objectmay include a biological micro-object.

43. The method of any one of embodiments 33-42, where the micro-objectmay include a hybridoma cell, a B cell or a T cell.

44. The method of any one of embodiments 33-43, where the first insitu-generated capture structure may be disposed at a first locationadjacent to a first wall of the sequestration pen.

45. The method of any one of embodiments 33-44, where the step ofcontacting the assay reagent or assay analyte with the biologicalproduct or the micro-object may further include forming a non-covalentcomplex.

46. The method of any one of embodiments 33-45, where the step ofdetecting the interaction may further include introducing a detectionreagent having a detectable label to the region proximal to the at leastfirst in situ-generated capture structure.

47. The method of embodiment 46, where the detectable label may beconfigured to be concentrated to the at least first in situ-generatedcapture structure when the assay reagent or assay analyte interacts withthe biological product or the micro-object.

48. The method of 46 or 47, where the detectable label of the detectionreagent is fluorescent, colorimetric, or luminescent.

49. The method of any one of embodiments 46-48, where the detectionreagent may include at least a first antibody.

50. The method of embodiment 49, where the antibody detection reagentmay further include a secondary antibody.

51. The method of any one of embodiments 46-48, where the detectionreagent may include an intercalating dye.

52. The method of any one of embodiments 46-48, where the detectionreagent may include an oligonucleotide.

53. The method of embodiment 46-53, where the step of detecting aninteraction of the assay reagent or assay analyte with the micro-objector the biological product may further include quantifying the amount ofdetectable label attached to the at least one in situ-generated capturestructure.

54. The method of any one of embodiments 33-53, where the step ofdetecting may include detecting a fluorescent signal from the at leastone in situ-generated capture structure.

55. The method of any one of embodiments 34-54, where the microfluidicdevice may further include a second in situ-generated capture structuredisposed within the flow region or the at least one sequestration pen,where the in situ-generated second capture structure may include asecond solidified polymer network, and further where the insitu-generated second solidified polymer network may include a secondassay reagent or assay analyte.

56. The method of embodiment 55, where each of the first and second insitu-generated capture structures may include a different assay reagentor assay analyte.

57. The method of embodiment 55 or 56, where the first and the second insitu-generated capture structures may be disposed within the flow regionor the sequestration pen at distinguishable locations.

58. The method of any one of embodiments 55-57, where the step ofdetecting may include detecting a first biological product of themicro-object and a second biological product of the micro-object, wherethe first biological product is different from the second biologicalproduct.

59. The method of any one of embodiments 55-58, where the step ofdetecting the interaction may further include introducing a firstdetection reagent and a second detection reagent to the region proximalto the first and second in situ-generated capture structures, where eachof the first and second detection reagents may include a detectablelabel.

60. The method of embodiment 59, where the detecting step may includeallowing each of the first and second detectable labels to becomenon-covalently attached to the respective first assay reagent or assayanalyte and the second assay reagent or assay analyte.

61. The method of embodiment 59 or 60, where the step of detecting theinteraction may further include detecting a first fluorescent signalfrom the first detectable label and a second fluorescent signal from thesecond detectable label.

62. The method of embodiment 61, where the first and the secondfluorescent signals may be spectrally distinct.

63. The method of embodiment 61 or 62, where the first and secondfluorescent signals may be distinguishable by position.

64. The method of any one of embodiments 61-63, further comprising thestep of quantifying the first and/or the second fluorescent signals.

65. The method of any one of embodiments 55-64, further including athird or more in situ-generated capture structure disposed within theflow region or the at least one sequestration pen, where each of thethird or more in situ-generated capture structures may include asolidified polymer network, and further where the solidified polymernetwork of each of the third or more solidified polymer networks mayinclude an assay reagent or assay agent.

66. The method of embodiment 65, where the step of detecting may furtherinclude detecting a first, a second, a third or more detectable signalsthat are distinct in location within the flow region or the at least onesequestration pen, detectably spectrally distinct from each other, or acombination thereof.

67. The method of any one of embodiments 33-66, where the step ofdisposing the micro-object within the microfluidic device in the regionproximal to the at least first in situ-generated capture structure mayinclude moving the micro-object using dielectrophoresis force.

68. The method of embodiment 67, where the dielectrophoresis force maybe optically actuated.

69. The method of any one of embodiments 33-68, where at least one innersurface of the microfluidic device may further include a conditionedsurface.

70. A kit including: a microfluidic device having an enclosure includinga substrate, microfluidic circuit material, and, optionally, a cover,the enclosure defining a flow region; and a functionalized pre-polymerthat can be controllably activated to form a solidified polymer network.

71. A kit including: a microfluidic device having an enclosure includinga substrate, microfluidic circuit material, and, optionally, a cover,the enclosure defining a flow region; and at least one in situ-generatedcapture structure disposed within enclosure, where the at least one insitu-generated capture structure includes a solidified polymer network.

72. The kit of embodiment 70 or 71, wherein the enclosure of themicrofluidic device further comprises at least one sequestration penfluidically connected to the flow region.

73. The kit of any one of embodiments 70-72, where the solidifiedpolymer network may include one or more functionalized sites.

74. The kit of any one of embodiments 70-73, further including an assayreagent or assay analyte.

75. The kit of embodiment 74, where the assay reagent or assay analytemay further include a functionalized assay reagent or assay analyte.

76. The kit of embodiment 75, where the functionalized assay reagent orassay analyte may include a moiety configured to associate or bind tothe one or more functionalized sites of the solidified polymer network.

77. The kit of any one of embodiments 74-76, where the solidifiedpolymer network may include the assay reagent or assay analyte.

78. The kit of embodiment 77, where the assay reagent or assay analytemay be covalently attached to the solidified polymer network.

79. The kit of embodiment 77, where the assay reagent or assay analytemay be non-covalently attached to the solidified polymer network.

80. The kit of embodiment 79, where the assay reagent or assay analytemay be non-covalently attached to the solidified polymer network via abiotin/streptavidin complex.

81. The kit of any one of embodiments 70-80, where the solidifiedpolymer network may include a synthetic polymer, a modified syntheticpolymer, or a biological polymer.

82. The kit of embodiment 81, where the synthetic polymer modificationsmay include size modification motifs, cleavage motifs, reactive terminalmoieties, and/or cell recognition motifs.

83. The kit of any one of embodiments 70-82, where the solidifiedpolymer network may include at least one of a polyethylene glycol,modified polyethylene glycol, polylactic acid (PLA), modified polylacticacid, polyglycolic acid (PGA), modified polyglycolic acid,polyacrylamide (PAM), modified polyacrylamide,poly-N-isopropylacrylamide (PNIPAm), modifiedpoly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinylalcohol, polyacrylic acid (PAA), modified polyacrylic acid,polycaprolactone (PCL), modified polycaprolactone, fibronectin, modifiedfibronectin, collagen, modified collagen, laminin, modified laminin,polysaccharide, modified polysaccharide, or a co-polymer in anycombination.

84. The kit of any one of embodiments, 70-83, wherein the solidifiedpolymer network includes a modified polyethylene glycol.

85. The kit of any one of embodiments 74-84, where the assay reagent orassay analyte may include a protein, a nucleic acid, an organicmolecule, or a saccharide.

86. The kit of any one of embodiments 74-85, where the assay reagent orassay analyte may include an antibody.

87. The kit of any one of embodiments 74-85, where the assay reagent orassay analyte may include an antigen.

88. The kit of any one of embodiments 74-85, where the assay reagent mayinclude a capture oligonucleotide.

89. The kit of any one of embodiments 70-88, further including adetection reagent.

90. The kit of embodiment 89, where the detection reagent may include adetectable label.

91. The kit of embodiment 90, where the detectable label of thedetection reagent may include a fluorescent, colorimetric, orluminescent label.

92. The kit of any one of embodiments 89-91, where the detection reagentmay include at least a first antibody.

93. The kit of any one of embodiments 89-91, where the detection reagentmay include an intercalating dye.

94. The kit of any one of embodiments 89-91, where the detection reagentmay include a FRET labeled oligonucleotide.

95. The kit of any one of embodiments 71-94, where the enclosure mayinclude at least first and second in situ-generated capture structuresdisposed therein, where the first in situ-generated capture structureincludes a first solidified polymer network, and where the second insitu-generated capture structure includes a second solidified polymernetwork.

96. The kit of embodiment 95, where the first and second situ-generatedcapture structures are disposed within the flow region.

97. The kit of embodiment 95, where the first and second insitu-generated capture structures are disposed within the at least onesequestration pen.

98. The kit of any one of embodiments 95-97, where a first insitu-generated capture structure and a second in situ-generated capturestructure may be disposed in different locations within the flow regionor sequestration pen.

99. The kit of embodiment 70, further comprising a second functionalizedpre-polymer that can be controllably activated to form a secondsolidified polymer network.

100. The kit of any one of embodiments 95-99, which further includes afirst assay reagent and second assay reagent, and further where thefirst assay reagent may be different from the second assay reagent.

101. The kit of embodiment 100, where the first solidified polymernetwork includes the first assay reagent and the second solidifiedpolymer network includes the second assay reagent.

102. The kit of any one of embodiments 95-101, further including a firstdetection reagent and a second detection reagent, where the firstdetection reagent may be different from the second detection reagent.

103. The kit of embodiment 102, where the first detection reagentincludes a first detectable label and the second detection reagentincludes a second detectable label, where the first detectable label andthe second detectable label may be spectrally distinct.

104. The kit of any one of embodiments 70-103, where the enclosure ofthe microfluidic device may further include a plurality of sequestrationpens.

105. The kit of embodiment 104, where each of the plurality ofsequestration pens may include at least one capture structure includinga solidified polymer network.

106. The kit of any one of embodiments 70-105, where at least one innersurface of the microfluidic device may further include a conditionedsurface.

107. A method of preparing a microfluidic device including at least afirst in situ-generated capture structure, including: providing themicrofluidic device, where the microfluidic device comprises anenclosure including a substrate and a microfluidic circuit material, theenclosure defining a flow region; introducing a first flowablefunctionalized pre-polymer into the flow region; and activatingsolidification of the first flowable functionalized pre-polymer at atleast one selected area of the enclosure, thereby forming the at least afirst in situ-generated capture structure therein.

108. The method of embodiment 107, where the enclosure further definesat least one sequestration pen.

109. The method of embodiment 107 or 108, where the first insitu-generated capture structure is formed in the flow region.

110. The method of embodiment 108, where the first in situ-generatedcapture structure is formed in the sequestration pen.

111. The method of any one of embodiments 107-110, where the at leastfirst in-situ generated capture structure may include a solidifiedpolymer network including one or more functionalized sites.

112. The method of embodiment 110, where the one or more functionalizedsites may include a biotin, avidin, or streptavidin moiety.

113. The method of embodiment 111 or 112, where the one or morefunctionalized sites may be covalently bound to at least one componentof the first flowable functionalized pre-polymer.

114. The method of any one of embodiments 107-113, further including astep of flowing a first volume of a first fluidic medium through theflow region of the microfluidic device, thereby diffusing unsolidifiedfirst flowable functionalized pre-polymer out of the flow region and,optionally, the at least one sequestration pen.

115. The method of any one of embodiments 107-114, further includingintroducing a first functionalized assay reagent or assay analyte to theat least first capture structure; and associating the firstfunctionalized assay reagent or assay analyte to the functionalizedsites of the solidified polymer network of the at least first capturestructure.

116. The method of embodiment 115, further including flowing a secondvolume of the first fluidic medium through the microfluidic device,thereby diffusing unassociated first functionalized assay reagent orassay analyte out of the flow region and, optionally, the at least onesequestration pen.

117. The method of embodiment 115 or 116, where the first functionalizedassay reagent or assay analyte may include an antibody, antigen, organicmolecule, or an oligonucleotide.

118. The method of embodiment 117, where the organic molecule of thefirst functionalized assay reagent or assay analyte may include asubstrate to an enzyme, an antigen, a cell surface marker, or acytokine.

119. The method of any one of embodiments 115-118, where the firstfunctionalized assay reagent or assay analyte may further include amoiety configured to associate the first functionalized assay reagent orassay analyte with the functionalized site of the solidified polymernetwork of the at least first capture structure.

120. The method of embodiment 119, where the moiety configured toassociate the first functionalized assay reagent or assay analyte mayinclude a biotin, avidin or streptavidin binding partner to thefunctionalized site of the solidified polymer network of the at leastfirst capture structure.

121. The method of any one of embodiments 115-120, where the firstfunctionalized assay reagent or assay analyte may be a first assayreagent.

122. The method of any one of embodiments 107-121, further including thestep of introducing a second functionalized assay reagent or assayanalyte.

123. The method of embodiment 122, where the second functionalized assayreagent or assay analyte may associate with second functionalized sitesof the solidified polymer network of the at least first capturestructure.

124. The method of embodiment 123, where the second functionalized assayreagent or assay analyte may be detectably differentiable from the firstfunctionalized assay reagent or assay analyte.

125. The method of embodiment 123, where the second functionalized assayreagent or assay analyte may associate with first functionalized siteson a second capture structure in the at least one sequestration pen.

126. The method of embodiment 125, further including introducing thesecond capture structure in the flow region or the at least onesequestration pen, where introducing the second capture structure mayinclude the steps of: introducing a second flowable functionalizedpre-polymer into the flow region; and activating solidification of thesecond flowable functionalized pre-polymer at at least a second selectedarea of the enclosure, thereby forming the second in situ-generatedcapture structure therein; and flowing yet another volume of the firstfluidic medium into the flow region of the microfluidic device.

127. The method of embodiment 126, further including introducing a thirdcapture structure into the flow region or the at least one sequestrationpen, where introducing the third capture structure may include:introducing a third flowable functionalized pre-polymer into the flowregion; and activating solidification of the third flowablefunctionalized pre-polymer at at least a third selected area of theenclosure, thereby forming the third in situ-generated capture structuretherein; and flowing yet another volume of the first fluidic medium intothe flow region of the microfluidic device.

128. The method of any one of embodiments 122-127, where the secondfunctionalized assay reagent or assay analyte may be different from thefirst functionalized assay reagent or assay analyte.

129. The method of any one of embodiments 126-128, where the firstflowable functionalized pre-polymer is different from the secondflowable functionalized pre-polymer.

130. The method of any one of embodiments 127-129, where each of thefirst, second, and third flowable functionalized pre-polymer may bedifferent from each other.

131. The method of any one of embodiments 127-130, further including thestep of introducing a third functionalized assay reagent or assayanalyte.

132. The method of embodiment 131, where the third functionalized assayreagent or assay analyte may associate with a first functionalized siteon the third capture structure in the at least one sequestration pen.

133. The method of embodiment 131 or 132, where the third functionalizedassay reagent or assay analyte may be different from the firstfunctionalized assay reagent or assay analyte and from the secondfunctionalized assay reagent or assay analyte.

134. The method of any one of embodiments 107-133, where at least oneinner surface of the microfluidic device may further include aconditioned surface.

In addition to any previously indicated modification, numerous othervariations and alternative arrangements may be devised by those skilledin the art without departing from the spirit and scope of thisdescription, and appended claims are intended to cover suchmodifications and arrangements. Thus, while the information has beendescribed above with particularity and detail in connection with what ispresently deemed to be the most practical and preferred aspects, it willbe apparent to those of ordinary skill in the art that numerousmodifications, including, but not limited to, form, function, manner ofoperation, and use may be made without departing from the principles andconcepts set forth herein. Also, as used herein, the examples andembodiments, in all respects, are meant to be illustrative only andshould not be construed to be limiting in any manner. Furthermore, wherereference is made herein to a list of elements (e.g., elements a, b, c),such reference is intended to include any one of the listed elements byitself, any combination of less than all of the listed elements, and/ora combination of all of the listed elements. As used herein, the termsa, an, and one may each be interchangeable with the terms at least oneand one or more. It should also be noted, that while the term step isused herein, that term may be used to simply draw attention to differentportions of the described methods and is not meant to delineate astarting point or a stopping point for any portion of the methods, or tobe limiting in any other way.

1. A microfluidic device comprising: an enclosure comprising a substrateand microfluidic circuit material, the enclosure defining a flow regionand at least one sequestration pen, each located within the enclosure;and at least one in situ-generated capture structure disposed within theat least one sequestration pen, wherein the at least one insitu-generated capture structure comprises a solidified polymer network.2. The microfluidic device of claim 1, wherein the solidified polymernetwork comprises one or more functionalized sites.
 3. The microfluidicdevice of claim 1 or 2, wherein the solidified polymer network comprisesan assay reagent or assay analyte.
 4. The microfluidic device of claim1, wherein the at least one sequestration pen comprises an isolationregion and a connection region, the connection region having a proximalopening to the flow region and a distal opening to the isolation region.5. The microfluidic device of claim 4, wherein the at least one insitu-generated capture structure is disposed within the isolation regionof the sequestration pen.
 6. The microfluidic device of claim 3, whereinthe assay reagent is non-covalently attached to the solidified polymernetwork.
 7. (canceled)
 8. The microfluidic device of claim 3, whereinthe assay reagent comprises a protein, a nucleic acid, an organicmolecule, and/or a saccharide.
 9. The microfluidic device of claim 8,wherein the assay reagent comprises an antibody or an antigen. 10.-12.(canceled)
 13. The microfluidic device of claim 1 or 2, wherein two ormore in situ-generated capture structures are disposed in the at leastone sequestration pen. 14.-16. (canceled)
 17. The microfluidic device ofclaim 1 or 2, wherein the solidified polymer network comprises aphotoinitiated polymer.
 18. The microfluidic device of claim 1 or 2,wherein the solidified polymer network comprises a synthetic polymer, amodified synthetic polymer, a biological polymer, or any combinationthereof. 19.-20. (canceled)
 21. The microfluidic device of claim 1 or 2,wherein the substrate is configured to generate dielectrophoresis (DEP)forces within the enclosure.
 22. (canceled)
 23. A method of assaying amicro-object in a microfluidic device comprising at least a first insitu-generated capture structure comprising: disposing a micro-objectwithin the microfluidic device in a region proximal to the at leastfirst in situ-generated capture structure comprising a solidifiedpolymer network, wherein the solidified polymer network comprises anassay reagent or assay analyte; contacting the assay reagent or assayanalyte with the micro-object or a biological product of themicro-object; and detecting an interaction of the assay reagent or assayanalyte with the micro-object or the biological product.
 24. The methodof claim 23, wherein the microfluidic device comprises: an enclosurecomprising a substrate and microfluidic circuit material, the enclosuredefining a flow region and at least one sequestration pen; and whereinthe at least first in situ-generated capture structure is disposedwithin the at least one sequestration pen.
 25. The method of claim 24,wherein the at least one sequestration pen comprises an isolation regionand a connection region, the connection region having a proximal openingto the flow region and a distal opening to the isolation region, andfurther wherein the at least first capture structure is disposed withinthe isolation region of the sequestration pen.
 26. (canceled)
 27. Themethod of claim 23, wherein the assay reagent or assay analyte isnon-covalently attached to the solidified polymer network.
 28. Themethod of claim 23, wherein the solidified polymer network comprises anassay reagent, and wherein the assay reagent comprises a protein. 29.The method of claim 28, wherein the assay reagent comprises an antigenor an antibody.
 30. The method of claim 23, wherein the biologicalproduct comprises a protein, an oligonucleotide, an organic molecule, ora saccharide.
 31. The method of claim 23, wherein the biological productof the micro-object is an antibody or an antigen. 32.-34. (canceled) 35.The method of claim 23, wherein the step of contacting the assay reagentor assay analyte with the biological product or the micro-object furthercomprises forming a non-covalent complex.
 36. The method of claim 23,wherein the step of detecting the interaction further comprisesintroducing a detection reagent having a detectable label to the regionproximal to the at least first in situ-generated capture structure. 37.The method of claim 36, wherein the detectable label is configured to beconcentrated to the at least first in situ-generated capture structurewhen the assay reagent or assay analyte interacts with the biologicalproduct or the micro-object. 38.-39. (canceled)
 40. The method of claim23, wherein the step of detecting comprises detecting a fluorescentsignal from the at least first in situ-generated capture structure. 41.(canceled)
 42. The method of claim 24, wherein the microfluidic devicefurther comprises a second in situ-generated capture structure disposedwithin the at least one sequestration pen, wherein the second insitu-generated capture structure comprises a second solidified polymernetwork and further wherein the second solidified polymer networkcomprises a second assay reagent or assay analyte, wherein each of thefirst and second in situ-generated capture structures comprises adifferent assay reagent or assay analyte, and wherein the step ofdetecting comprises detecting a first biological product of themicro-object and a second biological product of the micro-object,wherein the first biological product is different from the secondbiological product. 43.-49. (canceled)
 50. The method of claim 23,wherein the step of disposing the micro-object within the microfluidicdevice in the region proximal to the at least first in situ-generatedcapture structure comprises moving the micro-object usingdielectrophoresis force.
 51. (canceled)
 52. A kit comprising amicrofluidic device comprising: an enclosure comprising a substrate andmicrofluidic circuit material, the enclosure defining a flow region andat least one sequestration pen; and a functionalized pre-polymer thatcan be controllably activated to form a solidified polymer network.53.-54. (canceled)
 55. The kit of claim 52, further comprising an assayreagent or assay analyte. 56.-74. (canceled)
 75. A method of preparing amicrofluidic device comprising at least a first in situ-generatedcapture structure, the method comprising: providing the microfluidicdevice, wherein the microfluidic device comprises an enclosurecomprising a substrate and microfluidic circuit material, the enclosuredefining a flow region and at least one sequestration pen fluidicallyconnected to the flow region; introducing a first flowablefunctionalized pre-polymer into the flow region; and activatingsolidification of the first flowable functionalized pre-polymer at atleast one selected area of the at least one sequestration pen, therebyforming the at least a first in situ-generated capture structuretherein. 76.-89. (canceled)